Substituted tetracycline compounds

ABSTRACT

The present invention pertains, at least in part, to novel substituted tetracycline compounds. These tetracycline compounds can be used to treat numerous tetracycline compound-responsive states, such as bacterial infections and neoplasms, as well as other known applications for tetracycline compounds such as blocking tetracycline efflux and modulation of gene expression.

RELATED APPLICATIONS

This patent application claims priority to U.S. Provisional PatentApplication Ser. No. 60/530,123, filed Dec. 16, 2003; U.S. ProvisionalPatent Application Ser. No. 60/525,287, filed Nov. 25, 2003; and U.S.Provisional Patent Application Ser. No. 60/486,017, filed Jul. 9, 2003,all of which are entitled “Substituted Tetracycline Compounds.” Theentire contents of each of the aforementioned applications are herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

The development of the tetracycline antibiotics was the direct result ofa systematic screening of soil specimens collected from many parts ofthe world for evidence of microorganisms capable of producingbacteriocidal and/or bacteriostatic compositions. The first of thesenovel compounds was introduced in 1948 under the name chlortetracycline.Two years later, oxytetracycline became available. The elucidation ofthe chemical structure of these compounds confirmed their similarity andfurnished the analytical basis for the production of a third member ofthis group in 1952, tetracycline. A new family of tetracyclinecompounds, without the ring-attached methyl group present in earliertetracyclines, was prepared in 1957 and became publicly available in1967; and minocycline was in use by 1972.

Recently, research efforts have focused on developing new tetracyclineantibiotic compositions effective under varying therapeutic conditionsand routes of administration. New tetracycline analogues have also beeninvestigated which may prove to be equal to or more effective than theoriginally introduced tetracycline compounds. Examples include U.S. Pat.Nos. 2,980,584; 2,990,331; 3,062,717; 3,165,531; 3,454,697; 3,557,280;3,674,859; 3,957,980; 4,018,889; 4,024,272; and 4,126,680. These patentsare representative of the range of pharmaceutically active tetracyclineand tetracycline analogue compositions.

Historically, soon after their initial development and introduction, thetetracyclines were found to be highly effective pharmacologicallyagainst rickettsiae; a number of gram-positive and gram-negativebacteria; and the agents responsible for lymphogranuloma venereum,inclusion conjunctivitis, and psittacosis. Hence, tetracyclines becameknown as “broad spectrum” antibiotics. With the subsequent establishmentof their in vitro antimicrobial activity, effectiveness in experimentalinfections, and pharmacological properties, the tetracyclines as a classrapidly became widely used for therapeutic purposes. However, thiswidespread use of tetracyclines for both major and minor illnesses anddiseases led directly to the emergence of resistance to theseantibiotics even among highly susceptible bacterial species bothcommensal and pathogenic (e.g., pneumococci and Salmonella). The rise oftetracycline-resistant organisms has resulted in a general decline inuse of tetracyclines and tetracycline analogue compositions asantibiotics of choice.

SUMMARY OF THE INVENTION

In one embodiment, the invention pertains to a 7,9-substitutedtetracycline compound of Formula I:

wherein:

X is CHC(R¹³Y′Y), CR^(6′)R⁶, S, NR⁶, or O;

R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, hydroxyl, halogen, orhydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ ethyl, perhalogenated alkenyl, substituted pyridinyl, pyrazinyl,furanyl, or pyrazolyl;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R⁹ —CH₂NR^(9a)R^(9b);

R^(9a) and R^(9b) are each independently hydrogen, alkyl, alkenyl orlinked to form a heterocycle;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.

In another embodiment, the invention pertains to a 9-substitutedtetracycline compound of formula II:

wherein:

X is CHC(R¹³Y′Y), CR^(6′)R⁶, S, NR⁶, or O;

R², R^(4′), R^(4″), R^(7′) and R^(7″) are each hydrogen, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrug moiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are independently hydrogen, methylene, absent, hydroxyl,halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is NR^(7′)R^(7″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R⁹ is —CH₂NR^(9a)R^(9b), or linked with R¹⁰ to form a furanyl ring;

R^(9a) is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,or heteroaromatic;

R^(9b) is hydrogen or alkyl;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts, esters and prodrugs thereof.

In another embodiment, the invention pertains to 7-substitutedtetracycline compounds of formula III:

wherein:

X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O;

R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is substituted or unsubstituted pyrazolyl, furanyl, thiophenyl, orthiazolyl;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R⁹ is hydrogen;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.

In another embodiment, the invention pertains to 8-substitutedtetracycline compound of formula IV:

wherein:

X is CHC(R¹³Y′Y), CR^(6′)R⁶, S, NR⁶, or O;

R², R^(4′), R^(4″), R^(7′) and R^(7″) are each hydrogen, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrug moiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are independently hydrogen, methylene, absent, hydroxyl,halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(7c))₀₋₁C(═W′)WR^(7a);

R⁸ is an aminomethyl substituted phenyl or substituted pyridinyl;

R⁹ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(9c)C(═Z′)ZR^(9a);

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(9a), R^(9b), R^(9c),R^(9d), R^(9e), and R^(8f) are each independently absent, hydrogen,acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

W is CR^(7d)R^(7e), S, O or NR^(7b);

W′ is O, NR^(7f), or S;

Z is CR^(9d)R^(9e), S, O or NR^(9b);

Z′ is O, NR^(9f), or S;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts, esters and prodrugs thereof.

In one embodiment, a 13-substituted tetracycline compound is of formulaV:

wherein:

R², R^(4′), R^(4″), R^(7′) and R^(7″) are each hydrogen, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrug moiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁷ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(c))₀₋₁C(═W′)WR^(7a);

R⁸ is substituted phenyl or substituted pyridinyl;

R⁹ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(9c)C(═Z′)ZR^(9a);

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(9a), R^(9b), R^(9e),R^(9d), R^(9e), and R^(8f) are each independently absent, hydrogen,acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

W is CR^(7d)R^(7e), S, O or NR^(7b);

W′ is O, NR^(7f), or S;

R¹³ is 4-alkyl substituted phenyl, and pharmaceutically acceptablesalts, esters and prodrugs thereof.

In another further embodiment, the invention pertains, at least in part,to methods for treating subjects for tetracycline responsive states byadministering to them an effective amount of a tetracycline compound ofthe invention, e.g., a compound of formula I, II, III, IV, V, or atetracycline compound otherwise described herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains, at least in part, to novel substitutedtetracycline compounds. These tetracycline compounds can be used totreat numerous tetracycline compound-responsive states, such asbacterial infections and neoplasms, as well as other known applicationsfor minocycline and tetracycline compounds in general, such as blockingtetracycline efflux and modulation of gene expression.

The term “tetracycline compound” includes many compounds with a similarring structure to tetracycline. Examples of tetracycline compoundsinclude: chlortetracycline, oxytetracycline, demeclocycline,methacycline, sancycline, chelocardin, rolitetracycline, lymecycline,apicycline; clomocycline, guamecycline, meglucycline, mepylcycline,penimepicycline, pipacycline, etamocycline, penimocycline, etc. Otherderivatives and analogues comprising a similar four ring structure arealso included (See Rogalski, “Chemical Modifications of Tetracyclines,”the entire contents of which are hereby incorporated herein byreference). Table 1 depicts tetracycline and several known othertetracycline derivatives.

TABLE 1

Other tetracycline compounds which may be modified using the methods ofthe invention include, but are not limited to,6-demethyl-6-deoxy-4-dedimethylaminotetracycline; tetracyclino-pyrazole;7-chloro-4-dedimethylaminotetracycline;4-hydroxy-4-dedimethylaminotetracycline;12α-deoxy-4-dedimethylaminotetracycline;5-hydroxy-6α-deoxy-4-dedimethylaminotetracycline;4-dedimethylamino-12α-deoxyanhythotetracycline;7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline;tetracyclinonitrile; 4-oxo-4-dedimethylaminotetracycline 4,6-hemiketal;4-oxo-11a C1-4-dedimethylaminotetracycline-4,6-hemiketal;5a,6-anhydro-4-hydrazon-4-dedimethylamino tetracycline;4-hydroxyimino-4-dedimethylamino tetracyclines;4-hydroxyimino-4-dedimethylamino 5a,6-anhydrotetracyclines;4-amino-4-dedimethylamino-5a,6 anhydrotetracycline;4-methylamino-4-dedimethylamino tetracycline;4-hydrazono-11a-chloro-6-deoxy-6-demethyl-6-methylene-4-dedimethylaminotetracycline; tetracycline quaternary ammonium compounds;anhydrotetracycline betaines; 4-hydroxy-6-methyl pretetramides; 4-ketotetracyclines; 5-keto tetracyclines; 5a,11a dehydro tetracyclines; 11aC1-6, 12 hemiketal tetracyclines; 11a C1-6-methylene tetracyclines; 6,13 diol tetracyclines; 6-benzylthiomethylene tetracyclines;7,11a-dichloro-6-fluoro-methyl-6-deoxy tetracyclines; 6-fluoro(α)-6-demethyl-6-deoxy tetracyclines; 6-fluoro (β)-6-demethyl-6-deoxytetracyclines; 6-α acetoxy-6-demethyl tetracyclines; 6-βacetoxy-6-demethyl tetracyclines; 7, 13-epithiotetracyclines;oxytetracyclines; pyrazolotetracyclines; 11a halogens of tetracyclines;12a formyl and other esters of tetracyclines; 5,12a esters oftetracyclines; 10,12a-diesters of tetracyclines; isotetracycline;12-a-deoxyanhydro tetracyclines;6-demethyl-12a-deoxy-7-chloroanhydrotetracyclines; B-nortetracyclines;7-methoxy-6-demethyl-6-deoxytetracyclines;6-demethyl-6-deoxy-5a-epitetracyclines; 8-hydroxy-6-demethyl-6-deoxytetracyclines; monardene; chromocycline; 5a methyl-6-demethyl-6-deoxytetracyclines; 6-oxa tetracyclines, and 6 thia tetracyclines.

1. 7,9-Substituted Tetracycline Compounds

The invention also pertains, at least in part to 7,9-substitutedtetracycline compounds.

The term “7,9-substituted tetracycline compounds” includes tetracyclinecompounds with substitution at the 7 and 9-positions. In one embodiment,the substitution at the 7- and 9-positions enhances the ability of thetetracycline compound to perform its intended function, e.g., treattetracycline responsive states. In an embodiment, the 7,9-substitutedtetracycline compound is 7,9-substituted tetracycline (e.g., wherein R⁴is NR^(4′)R^(4″); R^(4′) and R^(4″) are methyl, R⁵ is hydrogen and X isCR⁶R^(6′), wherein R⁶ is methyl and R^(6′) is hydroxy); 7,9-substituteddoxycycline (e.g., wherein R⁴ is NR^(4′)R^(4″); R^(4′) and R^(4″) aremethyl, R⁵ is hydroxyl and X is CR⁶R^(6′), wherein R⁶ is methyl andR^(6′) is hydrogen); or 7, 9-substituted sancycline (wherein R⁴ isNR^(4′)R^(4″); R^(4′) and R^(4″) are methyl; R⁵ is hydrogen and X isCR⁶R^(6′) wherein R⁶ and R^(6′) are hydrogen atoms. In an embodiment,the substitution at the 7 position of the 7, 9-substituted tetracyclinecompound is not chlorine or trimethylamino. In one embodiment, R⁴ ishydrogen.

In one embodiment, the invention pertains to 7,9-substitutedtetracycline compounds of Formula I:

wherein:

X is CHC(R¹³Y′Y), CR^(6′)R⁶, S, NR⁶, or O;

R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, hydroxyl, halogen, orhydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is ethyl, perhalogenated alkenyl, substituted pyridinyl, pyrazinyl,furanyl, or pyrazolyl;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R⁹ is —CH₂NR^(9a)R^(9b);

R^(9a) and R^(9b) are each independently hydrogen, alkyl, alkenyl orlinked to form a heterocycle;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof, provided that R⁷ and R⁹ arenot both unsubstituted phenyl.

In a further embodiment, X is CR⁶R^(6′); R², R^(2′), R⁶, R^(6′), R⁸,R¹⁰, R¹¹, and R¹² are each hydrogen; R⁴ is NR^(4′)R^(4″); R^(4′) andR^(4″) are lower alkyl; and R⁵ is hydroxy or hydrogen. In anotherfurther embodiment, R^(4′) and R^(4″) are each methyl and R⁵ ishydrogen.

In an embodiment, R⁷ is ethyl and R^(9a) is alkyl and R^(9b) is alkenyl.In another embodiment, R⁷ is substituted pyrazinyl Examples of possiblesubstituents include halogens, such as fluorine. In another embodiment,R^(9a) is alkyl and R^(9b) is alkenyl. In another further embodiment,R^(9a) and R^(9b) are linked to form a heterocycle. In a furtherembodiment, the linked heterocycle is substituted piperidinyl. In afurther embodiment, the piperdinyl is substituted with one or morefluorines or halogenated alkyl groups, e.g., at the 2, 3, 4, or 5position. In another embodiment, the R⁹ moiety is(4′trifluoromethyl-piperdin-1-yl) methyl, (4′,4′-difluoro-piperdin-1-yl)methyl, or (4′-fluoropiperdin-1-yl) methyl.

In another embodiment, R^(9a) is hydrogen and R^(9b) is alkyl. Otherexamples of compounds include those wherein R⁷ is furanyl, and R^(9a) ishydrogen or alkyl and R^(9b) is alkenyl, e.g., 1, 2, 2-trifluoroethenyl.

In another embodiment, R^(9a) is hydrogen or alkyl and R^(9b) isalkenyl. In another embodiment, R⁷ is pyrazolyl and R^(9a) is hydrogenor alkyl and R^(9b) is alkenyl or alkyl.

In a further embodiment, the invention pertains to tetracyclinecompounds selected from the group consisting of:

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

2. 9-Substituted Tetracycline Compounds

In another embodiment, the invention pertains to 9-substitutedtetracycline compounds.

The term “9-substituted tetracycline compounds” includes tetracyclinecompounds with substitution at the 9 position. In one embodiment, thesubstitution at the 9-position enhances the ability of the tetracyclinecompound to perform its intended function, e.g., treat tetracyclineresponsive states. In an embodiment, the 9-substituted tetracyclinecompound is 9-substituted tetracycline (e.g., wherein R⁴ isNR^(4′)R^(4″), R^(4′) and R^(4″) are methyl, R⁵ is hydrogen and X isCR⁶R^(6′), wherein R⁶ is methyl and R^(6′) is hydroxy, and R⁷ ishydrogen); 9-substituted doxycycline (e.g., wherein R⁴ is NR^(4′)R^(4″),R^(4′) and R^(4″) are methyl, R⁵ is hydroxyl and X is CR⁶R^(6′), whereinR⁶ is methyl and R^(6′) is hydrogen, and R⁷ is hydrogen); 9-substitutedminocycline (wherein R⁴ is NR^(4′)R^(4″), R^(4′) and R^(4″) are methyl;R⁵ is hydrogen and X is CR⁶R^(6′) wherein R⁶ and R^(6′) are hydrogenatoms, and R⁷ is dimethylamino); 9-substituted 4-dedimethylaminotetracycline compound, wherein X is CR⁶R^(6′), R⁴, R⁵, R^(6′), R⁶, andR⁷ are hydrogen; and 9-substituted sancycline (wherein R⁴ isNR^(4′)R^(4″), R^(4′) and R^(4″) are methyl; R⁵ and R⁷ are hydrogen andX is CR⁶R^(6′) wherein R⁶ and R^(6′) are hydrogen atoms).

In another embodiment, the invention pertains to tetracycline compoundsof formula II:

wherein:

X is CHC(R¹³Y′Y), CR^(6′)R⁶, S, NR⁶, or O;

R², R^(4′), R^(4″), R^(7′) and R⁷ are each hydrogen, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrug moiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are independently hydrogen, methylene, absent, hydroxyl,halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is NR^(7′)R^(7″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R⁹ is —CH₂NR^(9a)R^(9b), or linked with R¹⁰ to form a furanyl ring;

R^(9a) is hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,or heteroaromatic;

R^(9b) is alkoxycarbonyl, arylaminocarbonyl, or aryloxycarbonyl;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts, esters and prodrugs thereof.

In a further embodiment, R⁴ is NR^(4′)R^(4″); X is CR⁶R^(6′); R⁷ isNR^(7′)R^(7″), R², R^(2′), R⁵, R⁶, R^(6′), R⁸, R⁹, R¹⁰, R¹¹, and R¹² areeach hydrogen; and, R^(4′), R^(4″), R^(7′), and R^(7″) are each loweralkyl. In another embodiment, R^(9a) is alkyl, alkenyl, or arylalkyl.Examples of R^(9b) include alkoxycarbonyl, alkaminocarbonyl,aryloxycarbonyl, and arylaminocarbonyl. In another embodiment, R^(9a)and R^(9b) are linked to form a heterocyle, e.g., a substituted orunsubstituted piperdinyl ring. In a further embodiment, the piperdinylis substituted with one or more fluorines or halogenated alkyl groups,e.g., at the 2, 3, 4, or 5 position. In another embodiment, the R⁹moiety is (4′trifluoromethyl-piperdin-1-yl) methyl,(4′,4′-difluoro-piperdin-1-yl) methyl, or (4′-fluoropiperdin-1-yl)methyl.

In another embodiment, R⁴ is NR^(4′)R^(4″), R^(4′) and R^(4″) aremethyl, R⁵ is hydroxyl and X is CR⁶R^(6′), wherein R⁶ is methyl andR^(6′) is hydrogen, and R⁷ is hydrogen In another embodiment, R^(9a) isalkyl, alkenyl, or arylalkyl. In a further embodiment, the piperdinyl issubstituted with one or more fluorines or halogenated alkyl groups,e.g., at the 2, 3, 4, or 5 position. In another embodiment, the R⁹moiety is (4′trifluoromethyl-piperdin-1-yl) methyl,(4′,4′-difluoro-piperdin-1-yl) methyl, or (4′-fluoropiperdin-1-yl)methyl.

In another further embodiment, R^(9a) is substituted alkyl. Examplesinclude alkoxy substituted alkyl (e.g., —(CH₂)₂—O—CH₃), alkenylsubstituted alkyl (e.g., —CH₂—CH═C(CH₃)₂, —CH₂—C(CH₃)═CHCH₃,—CH₂—CH═CH-phenyl, etc.), heterocyclic substituted alkyl (e.g.,—CH₂-furanyl, —CH₂—CH═CH-furanyl, —CH₂-pyridinyl, optionallysubstituted), cyano substituted alkyl (e.g., (CH₂)₂—CN, etc.), alkynylsubstituted alkyl (e.g., —(CH₂)₂—C≡CH, etc.), halogen substituted alkyl(e.g., (CH₂)₂—CF₃, (CH₂)₃—CF₃, —CH₂—CF₃, —CH₂—CH₂F, etc.), amidosubstituted alkyl (e.g., —CH₂—C(═O)—N(CH₃)₂, —CH₂—C(═O)—NH₂, etc.),carbonyl substituted alkyl (e.g., CH₂—C(═O)—CH₃, —CH₂—C(═O)—C(CH₃)₃,etc.), hydroxy substituted alkyl (e.g., (CH₂—CH(OH)—CH₃,—CH₂—C(OH)(CH₃)₂, etc.), —CH₂—C(═N—O—CH₃)—CH₃, cycloalkyl (e.g.,adamantyl, etc.).

In another embodiment, R^(9a) is substituted or unsubstituted benzyl. Ina further embodiment, R^(9a) is substituted with one or more fluorines(e.g., at the 2, 3, 4, 5, or 6 positions).

In a further embodiment, R^(9b) is hydrogen, substituted orunsubstituted alkyl (e.g., methyl, ethyl, —CH₂—CH═CH-furanyl,—CH₂—CH═C(CH₃)₂, —(CH₂)₃—CF₃, —(CH₂)₂—CH₂F, —CH₂—CH₂F, —(CH₂)₂—CF₃,—CH₂—CF₃, etc.).

In another further embodiment, R^(9a) and R^(9b) may be linked to form apyrrolidinyl, piperazinyl, piperidinyl, pyrazinyl, azapanyl,thiomorpholinyl, morpholinyl, tetrahydroquinolinyl, or adecahydroquinolinyl ring. The ring maybe substituted with one or morefluorines at the 2, 3, 4, or 5 position. The ring may also besubstituted with one or more fluorinated alkyl groups (e.g., CH₂F,—CHF₂, CF₃, etc.), cyano groups, hydroxy groups, alkyl groups (e.g.,methyl, ethyl, spiro-cyclohexyl, t-butyl, etc.), heterocyclic (e.g.,optionally substituted morpholinyl), thiol groups, alkoxy groups,alkyloxycarbonyl groups, carbonyl groups (optionally bonded directly toan atom in the ring), and exocyclic and endocyclic double bonds. In oneembodiment, the ring is substituted with a ═CF₂ group. The ring may alsobe linked to a —O—(CH₂)₂—O— group which maybe attached to thepyrollidinyl or piperidinyl ring through one carbons or through twoadjacent carbons.

When R⁹ is linked to R¹⁰ to form a furanyl ring, the ring can be furthersubstituted, e.g., with phenyl or other substituents which allow thecompound of the invention to perform its intended function.

In a further embodiment, the tetracycline compound is selected from thegroup consisting of:

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

3. 7-Substituted Tetracycline Compounds

In one embodiment, the invention pertains to novel 7-substitutedtetracycline compounds.

The term “7-substituted tetracycline compounds” includes tetracyclinecompounds with substitution at the 7 position. In one embodiment, thesubstitution at the 7-position enhances the ability of the tetracyclinecompound to perform its intended function, e.g., treat tetracyclineresponsive states. In an embodiment, the 7-substituted tetracyclinecompound is 7-substituted tetracycline (e.g., wherein R⁴ isNR^(4′)R^(4″), R^(4′) and R^(4″) are methyl, R⁵ is hydrogen and X isCR⁶R^(6′), wherein R⁶ is methyl and R^(6′) is hydroxy); 7-substituteddoxycycline (e.g., wherein R⁴ is NR^(4′)R^(4″), R^(4′) and R^(4″) aremethyl, R⁵ is hydroxyl and X is CR⁶R^(6′), wherein R⁶ is methyl andR^(6′) is hydrogen); 7-substituted tetracycline compound, wherein X isCR⁶R^(6′), R⁴, R⁵, R^(6′), and R⁶ are hydrogen; or 7-substitutedsancycline (wherein R⁴ is NR^(4′)R^(4″), R^(4′) and R^(4″) are methyl;R⁵ is hydrogen and X is CR⁶R^(6′) wherein R⁶ and R^(6′) are hydrogenatoms).

The invention pertains, at least in part, to 7-substituted tetracyclinecompound of Formula III:

wherein:

X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O;

R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is substituted or unsubstituted pyrazolyl, furanyl, thiophenyl, orthiazolyl;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R⁹ is hydrogen;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.

In a further embodiment, R⁴ is NR^(4′)R^(4″); X is CR⁶R^(6′), R²,R^(2′), R⁵, R⁶, R^(6′), R⁸, R⁹, R¹⁰, R¹¹, and R¹² are each hydrogen;and, R^(4′), and R⁴ are each lower alkyl, e.g., methyl.

In one embodiment, the tetracycline compound is a doxycycline compoundand R⁷ is substituted or unsubstituted aminomethyl (e.g.,—CH₂NR^(7a)R^(7b)).

In one embodiment, R⁷ is substituted (e.g., N-alkyl substituted) orunsubstituted pyrazolyl. In another embodiment, R⁷ is diethyl amino. Inanother, R⁷ is substituted amino methyl. In a further embodiment, thesubstituted aminomethyl is substituted with a pentyl group (e.g.,—CH₂—C(CH₃)₃), two methyl groups, or fluorinated alkyl (e.g.,fluorinated propyl, e.g., —CH₂—CH₂—CF₃).

In another embodiment, R⁷ is substituted phenyl. In a furtherembodiment, R⁷ is phenyl substituted at the 5 position (of the phenylring) with an alkyl substituted amino methyl group (e.g., (—CH₂—N(CH₃)₂,—CH₂—NH—CH(CH₃)₂, —CH₂—N(CH₃)—CH(CH₃)₂, —CH₂—N-piperdinyl), —CH₂NH—CH₃,—CH₂—NH-cyclopropyl, CH₂—NH-t-butyl, —CH₂—N(CH₃)-benzyl,—CH₂—N(CH₃)—CH₂—CH═CH₂, CH₂—NH—(CH₂)₂—CF₃, CH₂—NH—CH₂—C(═O)—NH₂, or—CH₂—NH-cyclohexyl,). In a further embodiment, the piperidine may besubstituted at its 4 position (e.g., with fluorine, methyl, etc.).

In another embodiment, when R⁷ is a phenyl substituted at the 5 positionwith an alkyl substituted amino methyl group, the phenyl may also besubstituted with a fluorine (e.g., at the 2, 3, 4, or 6 position) or analkoxy (e.g., methoxy group) at the 2, 3, 4, or 6 position.

In another embodiment, R⁷ is phenyl with a 2-position amino alkylsubstituent. In a further embodiment, the substituent isdialkylaminomethyl (e.g., dimethylaminomethyl, —CH₂—N-piperazinyl). In afurther embodiment, the piperazine is substituted with one or morefluorine or methyl groups. In another further embodiment, the phenyl R⁷is further substituted at the 3, 4, 5, or 6 position with a methoxygroup. In another embodiment, the phenyl is linked to a methylene dioxygroup through its 4 and 5 positions.

In another embodiment, R⁷ is phenyl with a 4-position amino alkyl (e.g.,aminomethyl) substituent. In a further embodiment, the aminoalkylsubstituent is —CH₂—NH—CH(CH₃)₂, —C(CH₃)—NH—(CH₂)₂—CH₂F,—CH₂—NH—CH₂-cyclohexenyl, —CH₂—N— piperidinyl, —CH₂—N(CH₃)—CH₂—CH═CH₂,or —CH₂—NH—(CH₂)₂—CF₃).

In another embodiment, R⁷ is phenyl substituted with a —C(═N—O—R)—R′group, wherein R and R′ are each alkyl. In a further embodiment, thesubstituent is at the 4-position of the phenyl ring. In anotherembodiment, R⁷ is phenyl substituted at the 4-position with analkoxyalkyl group (—CH₂—O—CH₃). In another embodiment, R⁷ is phenylsubstituted with an alkylcarbonylamino group.

In another embodiment, R⁷ is substituted furanyl. In a furtherembodiment, the furanyl is attached at the 2-position of the furanylring. In a further embodiment, the furanyl is substituted with an aminoalkyl, e.g., aminomethyl group at its 5-position. Examples ofaminomethyl groups include: —CH₂N(CH₃)—CH₂—C₆H₅, —CH₂—N(CH₃)—CH₂—CH═CH₂,—CH₂—N(CH₃)—CH(CH₃)₂, or —CH₂—N-piperidinyl. In another embodiment, thefuranyl is substituted at the 3-position, e.g., with an aminoalkylsubstituent. Examples of such substituents include —CH₂—N(CH₃)₂,—CH₂—N-piperidinyl

In another embodiment, R⁷ is substituted furanyl attached at its3-position. In a further embodiment, the furanyl is substituted with anaminoalkyl substituent. In another further embodiment, the aminoalkylsubstituent is —CH₂—N-piperazinyl or —CH₂—N—(CH₃)₂.

In another embodiment, R⁷ is substituted or unsubstituted thiophenyl. Ina further embodiment R⁷ is substituted with an aminoalkyl moiety. Inanother further embodiment, the aminoalkyl moiety is —CH₂—N—(CH₃)₂.

In another further embodiment, R⁷ is substituted pyridinyl. In a furtherembodiment, R⁷ is attached to the phenyl ring at its 3-position. Inanother further embodiment, it is substituted with a aminoalkyl moietyat its 5-position. Examples of aminoalkyl moieties include—CH₂—N—(CH₃)₂, —CH₂—N-piperidinyl, —CH₂—N(CH₃)—CH₂—CH═CH₂, or—CH₂—N(CH₃)—CH(CH₃)₂.

In another further embodiment, R⁷ is alkylcarbonylaminoalkyl. In anotherfurther embodiment, R⁷ is —CH₂—NH—C(═O)—CH₃.

In another further embodiment, R⁷ is amino substituted alkenyl. Inanother further embodiment, R⁷ is —CH═CH—CH₂—N(CH₃)₂ or—CH═CH—CH₂—N-piperidinyl. In another embodiment, R⁷ is amino substitutedalkynyl (e.g., —C≡C—CH₂—N(CH₃)—(CH₂)₂—CF₃ or —C≡C—(CH₂)₂—N-piperidinyl.

In another further embodiment, R⁷ is substituted —CH₂—N-piperidinyl. Incertain embodiments, the piperidinyl is substituted with one or morefluorines, e.g., at the 4-position of the piperidine ring.

In another embodiment, the R⁷ substitutent is alkylaminocarbonyl. In afurther embodiment, the substituent is —C(═O)—NH—(CH₂)₂—N(CH₃)₂.

In another further embodiment, the R⁷ substituent is aminoalkylcarbonyl.In a further embodiment, the substituent is —C(═O)—CH₂—N(CH₃)₂,—C(═O)—CH₂—NH—(CH₂)₂—OCH₃, —C(═O)—CH₂—N-piperidinyl and—C(═O)—CH₂—N-pyrollidinyl.

In another further embodiment, the R⁷ substituent is N-piperdinylsubstituted alkyl. In a further embodiment, the R⁷ substituent is—(CH₂)₄—N-piperdinyl or —(CH₂)₂—N-piperdinyl.

In another embodiment, the R⁷ substituted is —(CH₂)₂—N(CH₃)₂ orC(═O)—CH₃.

In another further embodiment, the R⁷ substituent isaminoalkyloxycarbonyl. Examples of aminoalkyloxycarbonyl substituentsinclude C(═O)—O—(CH₂)₂—N-piperdinyl and —C(═O)—O—(CH₂)₂—N(CH₃)₂.

In a further embodiment, the compounds of the invention are:

and pharmaceutically acceptable esters, prodrugs, and salts thereof.

4. 8-Substituted Tetracycline Compounds

The invention also pertains, at least in part to 8-substitutedtetracycline compounds.

The term “8-substituted tetracycline compounds” includes tetracyclinecompounds with substitution at the 8-position. In one embodiment, thesubstitution at the 8-position enhances the ability of the tetracyclinecompound to perform its intended function, e.g., treat tetracyclineresponsive states. In an embodiment, the 8-substituted tetracyclinecompound is 8-substituted tetracycline (e.g., wherein R⁴ isNR^(4′)R^(4″); R^(4′) and R^(4″) are methyl, R⁵ is hydrogen and X isCR⁶R^(6′), wherein R⁶ is methyl and R^(6′) is hydroxy); 8-substituteddoxycycline (e.g., wherein R⁴ is NR^(4′)R^(4″); R^(4′) and R^(4″) aremethyl, R⁵ is hydroxyl and X is CR⁶R^(6′), wherein R⁶ is methyl andR^(6′) is hydrogen); or 8-substituted sancycline (wherein R⁴ isNR^(4′)R^(4″); R^(4′) and are methyl; R⁵ is hydrogen and X is CR⁶R^(6′)wherein R⁶ and R^(6′) are hydrogen atoms. In an embodiment, thesubstitution at the 7 position of the 8-substituted tetracyclinecompound is not chlorine or trimethylamino. In one embodiment, R⁴ ishydrogen.

In one embodiment, the 8-substituted tetracycline compound is of formulaIV:

wherein:

X is CHC(R¹³Y′Y), CR^(6′)R⁶, S, NR⁶, or O;

R², R^(4′), R^(4″), R^(7′) and R^(7″) are each hydrogen, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrug moiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are independently hydrogen, methylene, absent, hydroxyl,halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(7c))₀₋₁C(═W′)WR^(7a);

R⁸ is substituted phenyl or substituted pyridinyl;

R⁹ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(9c)C(═Z′)ZR^(9a);

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(9a), R^(9b), R^(9c),R^(9d), R^(9e), and R^(8f) are each independently absent, hydrogen,acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

W is CR^(7d)R^(7e), S, O or NR^(7b);

W′ is O, NR^(7f), or S;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts, esters and prodrugs thereof.

In a further embodiment, the invention pertains to compounds wherein Xis CR⁶R^(6′); R², R^(2′), R⁶, R^(6′), R⁸, R¹⁰, R¹¹; and R¹² are eachhydrogen; R⁴ is NR^(4′)R^(4″); R^(4′) and R^(4″) are lower alkyl; and R⁵is hydroxy or hydrogen.

In a further embodiment, R⁸ is substituted phenyl, e.g., o-substitutedphenyl, e.g., aminomethyl substituted phenyl. In a further embodiment,the 8-substituted tetracycline compound is:

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

In another further embodiment, R⁸ is substituted pyridinyl, e.g.,halo-substituted pyridinyl, e.g., 6-fluoro-pyrindin-3-yl. In a furtherembodiment, R⁹ is amino. In yet a further embodiment, the 8-substitutedtetracycline compound is:

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

5. 13-Substituted Methacycline Compounds

In one embodiment, a 13-substituted tetracycline compound is of formulaV:

wherein:

R², R^(4′), R^(4″), R^(7′) and R^(7″) are each hydrogen, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrug moiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁷ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃(NR^(7c))₀₋₁C(═W′)WR^(7a);

R⁸ is substituted phenyl or substituted pyridinyl;

R⁹ is hydrogen, hydroxyl, halogen, thiol, nitro, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,arylalkyl, amino, arylalkenyl, arylalkynyl, acyl, aminoalkyl,heterocyclic, thionitroso, or —(CH₂)₀₋₃NR^(9c)C(═Z′)ZR^(9a);

R^(7a), R^(7b), R^(7c), R^(7d), R^(7e), R^(7f), R^(9a), R^(9b), R^(9c),R^(9d), R^(9e), and R^(8f) are each independently absent, hydrogen,acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety;

W is CR^(7d)R^(7e), S, O or NR^(7b);

W′ is O, NR^(7f), or S;

R¹³ is 4-alkyl substituted phenyl, and pharmaceutically acceptablesalts, esters and prodrugs thereof.

In a further embodiment, the invention pertains to compounds wherein R²,R^(2′), R⁸, R¹⁰, R¹¹, and R¹² are each hydrogen; R⁴ is NR^(4′)R^(4″);R^(4′) and R^(4″) are lower alkyl; and R⁵ is hydroxy or hydrogen.

In a further embodiment, the phenyl R¹³ group is substituted with anaminomethyl substituent. In another further embodiment, the aminomethylsubstituent is dimethylaminomethyl. In another further embodiment, theinvention pertains to compounds of the formula:

and pharmaceutically acceptable salts, esters, and prodrugs thereof.

In one embodiment, the tetracycline compounds of the invention do notinclude those described in U.S. Ser. Nos. 09/660,598, 09/823,884,09/852,908, 10/819,343, 10/820,456, 09/894,805, 09/895,796, 09/895,812,09/895,797, 09/895,857, 10/097,634, 10/759,484, 10/337,914, 10/636,437,10/752,378, or 10/740,961. The entire contents of each of theseapplications are hereby incorporated herein in their entirety.

6. Methods for Synthesizing Tetracycline Compounds of the Invention

The tetracycline compounds of this invention can be synthesized usingthe methods described in the Schemes and/or by other techniques known tothose of ordinary skill in the art.

The substituted tetracycline compounds of the invention can besynthesized using the methods described in the following schemes and byusing art recognized techniques. All novel substituted tetracyclinecompounds described herein are included in the invention as compounds.

9- and 7-substituted tetracyclines can be synthesized by the methodshown in Scheme 1. As shown in Scheme 1, 9- and 7-substitutedtetracycline compounds can be synthesized by treating a tetracyclinecompound (e.g., doxycycline, 1A), with sulfuric acid and sodium nitrate.The resulting product is a mixture of the 7-nitro and 9-nitro isomers(1B and 1C, respectively). The 7-nitro (1B) and 9-nitro (1C) derivativesare treated by hydrogenation using hydrogen gas and a platinum catalystto yield amines 1D and 1E. The isomers are separated at this time byconventional methods. To synthesize 7- or 9-substituted alkenylderivatives, the 7- or 9-amino tetracycline compound (1E and 1F,respectively) is treated with HONO, to yield the diazonium salt (1G and1H). The salt (1G and 1H) is treated with an appropriate reactivereagent to yield the desired compound (e.g., in Scheme 1,7-cyclopent-1-enyl doxycycline (1H) and 9-cyclopent-1-enyl doxycycline(1I)).

As shown in Scheme 2, tetracycline compounds of the invention wherein R⁷is a carbamate or a urea derivative can be synthesized using thefollowing protocol. Sancycline (2A) is treated with NaNO₂ under acidicconditions forming 7-nitro sancycline (2B) in a mixture of positionalisomers. 7-nitrosancycline (2B) is then treated with H₂ gas and aplatinum catalyst to form the 7-amino sancycline derivative (2C). Toform the urea derivative (2E), isocyanate (2D) is reacted with the7-amino sancycline derivative (2C). To form the carbamate (2G), theappropriate acid chloride ester (2F) is reacted with 2C.

As shown in Scheme 3, tetracycline compounds of the invention, whereinR⁷ is a heterocyclic (i.e. thiazole) substituted amino group can besynthesized using the above protocol. 7-amino sancycline (3A) is reactedwith Fmoc-isothiocyanate (3B) to produce the protected thiourea (3C).The protected thiourea (3C) is then deprotected yielding the activesancycline thiourea (3D) compound. The sancycline thiourea (3D) isreacted with an α-haloketone (3E) to produce a thiazole substituted7-amino sancycline (3F).

7-alkenyl tetracycline compounds, such as 7-alkynyl sancycline (4A) and7-alkenyl sancycline (4B), can be hydrogenated to form 7-alkylsubstituted tetracycline compounds (e.g., 7-alkyl sancycline, 4C).Scheme 4 depicts the selective hydrogenation of the 7-position double ortriple bond, in saturated methanol and hydrochloric acid solution with apalladium/carbon catalyst under pressure, to yield the product.

In Scheme 5, a general synthetic scheme for synthesizing 7-position arylderivatives is shown. A Suzuki coupling of an aryl boronic acid with aniodosancycline compound is shown. An iodo sancycline compound (5B) canbe synthesized from sancycline by treating sancycline (5A) with at leastone equivalent N-iodosuccinimide (NIS) under acidic conditions. Thereaction is quenched, and the resulting 7-iodo sancycline (5B) can thenbe purified using standard techniques known in the art. To form the arylderivative, 7-iodo sancycline (5B) is treated with an aqueous base(e.g., Na₂CO₃) and an appropriate boronic acid (5C) and under an inertatmosphere. The reaction is catalyzed with a palladium catalyst (e.g.,Pd(OAc)₂). The product (5D) can be purified by methods known in the art(such as HPLC). Other 7-aryl, alkenyl, and alkynyl tetracyclinecompounds can be synthesized using similar protocols.

The 7-substituted tetracycline compounds of the invention can also besynthesized using Stille cross couplings. Stille cross couplings can beperformed using an appropriate tin reagent (e.g., R-SnBu₃) and ahalogenated tetracycline compound, (e.g., 7-iodosancycline). The tinreagent and the iodosancycline compound can be treated with a palladiumcatalyst (e.g., Pd(PPh₃)₂Cl₂ or Pd(AsPh₃)₂Cl₂) and, optionally, with anadditional copper salt, e.g., CuI. The resulting compound can then bepurified using techniques known in the art.

The compounds of the invention can also be synthesized using Heck-typecross coupling reactions. As shown in Scheme 6, Heck-typecross-couplings can be performed by suspending a halogenatedtetracycline compound (e.g., 7-iodosancycline, 6A) and an appropriatepalladium or other transition metal catalyst (e.g., Pd(OAc)₂ and CuI) inan appropriate solvent (e.g., degassed acetonitrile). The substrate, areactive alkene (6B) or alkyne (6D), and triethylamine are then addedand the mixture is heated for several hours, before being cooled to roomtemperature. The resulting 7-substituted alkenyl (6C) or 7-substitutedalkynyl (6E) tetracycline compound can then be purified using techniquesknown in the art.

To prepare 7-(2′-Chloro-alkenyl)-tetracycline compounds, the appropriate7-(alkynyl)-sancycline (7A) is dissolved in saturated methanol andhydrochloric acid and stirred. The solvent is then removed to yield theproduct (7B).

As depicted in Scheme 8, 5-esters of 9-substituted tetracyclinecompounds can be formed by dissolving the 9-substituted compounds (8A)in strong acid (e.g. HF, methanesulphonic acid, andtrifluoromethanesulfonic acid) and adding the appropriate carboxylicacid to yield the corresponding esters (8B).

As shown in Scheme 9 below, 7 and 9 aminomethyl tetracyclines may besynthesized using reagents such as hydroxymethyl-carbamic acid benzylester.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.The term alkyl further includes alkyl groups, which can further includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkyl has 6 or fewer carbon atoms in itsbackbone (e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain), andmore preferably 4 or fewer. Likewise, preferred cycloalkyls have from3-8 carbon atoms in their ring structure, and more preferably have 5 or6 carbons in the ring structure. The term C₁-C₆ includes alkyl groupscontaining 1 to 6 carbon atoms.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “arylalkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl” also includes the side chains of natural andunnatural amino acids.

The term “aryl” includes groups, including 5- and 6-membered single-ringaromatic groups that may include from zero to four heteroatoms, forexample, benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminoacarbonyl, arylalkyl aminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, arylalkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings which are not aromatic so as to form apolycycle (e.g., tetralin).

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

For example, the term “alkenyl” includes straight-chain alkenyl groups(e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups,cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl,cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substitutedcycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenylgroups. The term alkenyl further includes alkenyl groups which includeoxygen, nitrogen, sulfur or phosphorous atoms replacing one or morecarbons of the hydrocarbon backbone. In certain embodiments, a straightchain or branched chain alkenyl group has 6 or fewer carbon atoms in itsbackbone (e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain).Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in theirring structure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₂-C₆ includes alkenyl groups containing 2 to 6carbon atoms.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls”, the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond.

For example, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkylor cycloalkenyl substituted alkynyl groups. The term alkynyl furtherincludes alkynyl groups which include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆for straight chain, C₃-C₆ for branched chain). The term C₂-C₆ includesalkynyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkynyl includes both “unsubstituted alkynyls” and“substituted alkynyls”, the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “acyl” includes compounds and moieties which contain the acylradical (CH₃CO—) or a carbonyl group. It includes substituted acylmoieties. The term “substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by for example, alkyl groups,alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “acylamino” includes moieties wherein an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “aroyl” includes compounds and moieties with an aryl orheteroaromatic moiety bound to a carbonyl group. Examples of aroylgroups include phenylcarboxy, naphthyl carboxy, etc.

The terms “alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties. Examples ofhalogen substituted alkoxy groups include, but are not limited to,fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,dichloromethoxy, trichloromethoxy, etc.

The term “amine” or “amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The termincludes “alkyl amino” which comprises groups and compounds wherein thenitrogen is bound to at least one additional alkyl group. The term“dialkyl amino” includes groups wherein the nitrogen atom is bound to atleast two additional alkyl groups. The term “arylamino” and“diarylamino” include groups wherein the nitrogen is bound to at leastone or two aryl groups, respectively. The term “alkylarylamino,”“alkylaminoaryl” or “arylaminoalkyl” refers to an amino group which isbound to at least one alkyl group and at least one aryl group. The term“alkaminoalkyl” refers to an alkyl, alkenyl, or alkynyl group bound to anitrogen atom which is also bound to an alkyl group.

The term “amide,” “amido” or “aminocarbonyl” includes compounds ormoieties which contain a nitrogen atom which is bound to the carbon of acarbonyl or a thiocarbonyl group. The term includes “alkaminocarbonyl”or “alkylaminocarbonyl” groups which include alkyl, alkenyl, aryl oralkynyl groups bound to an amino group bound to a carbonyl group. Itincludes arylaminocarbonyl and arylcarbonylamino groups which includearyl or heteroaryl moieties bound to an amino group which is bound tothe carbon of a carbonyl or thiocarbonyl group. The terms“alkylaminocarbonyl,” “alkenylaminocarbonyl,” “alkynylaminocarbonyl,”“arylaminocarbonyl,” “alkylcarbonylamino,” “alkenylcarbonylamino,”“alkynylcarbonylamino,” and “arylcarbonylamino” are included in term“amide.” Amides also include urea groups (aminocarbonylamino) andcarbamates (oxycarbonylamino).

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom. Thecarbonyl can be further substituted with any moiety which allows thecompounds of the invention to perform its intended function. Forexample, carbonyl moieties may be substituted with alkyls, alkenyls,alkynyls, aryls, alkoxy, aminos, etc. Examples of moieties which containa carbonyl include aldehydes, ketones, carboxylic acids, amides, esters,anhydrides, etc.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “ester” includes compounds and moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are asdefined above.

The term “thioether” includes compounds and moieties which contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom which is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomwhich is covalently bonded to an alkynyl group.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings, e.g., the rings are “fused rings”. Rings that arejoined through non-adjacent atoms are termed “bridged” rings. Each ofthe rings of the polycycle can be substituted with such substituents asdescribed above, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, alkylaminoacarbonyl,arylalkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,arylcarbonyl, arylalkyl carbonyl, alkenylcarbonyl, aminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amido, amino (including alkyl amino, dialkylamino, arylamino,diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or anaromatic or heteroaromatic moiety.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur andphosphorus.

The term “prodrug moiety” includes moieties which can be metabolized invivo to a hydroxyl group and moieties which may advantageously remainesterified in vivo. Preferably, the prodrugs moieties are metabolized invivo by esterases or by other mechanisms to hydroxyl groups or otheradvantageous groups. Examples of prodrugs and their uses are well knownin the art (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J.Pharm. Sci. 66:1-19). The prodrugs can be prepared in situ during thefinal isolation and purification of the compounds, or by separatelyreacting the purified compound in its free acid form or hydroxyl with asuitable esterifying agent. Hydroxyl groups can be converted into estersvia treatment with a carboxylic acid. Examples of prodrug moietiesinclude substituted and unsubstituted, branch or unbranched lower alkylester moieties, (e.g., propionic acid esters), lower alkenyl esters,di-lower alkylamino lower-alkyl esters (e.g., dimethylaminoethyl ester),acylamino lower alkyl esters (e.g., acetyloxymethyl ester), acyloxylower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenylester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g.,with methyl, halo, or methoxy substituents) aryl and aryl-lower alkylesters, amides, lower-alkyl amides, di-lower alkyl amides, and hydroxyamides. Preferred prodrug moieties are propionoic acid esters and acylesters.

It will be noted that the structure of some of the tetracyclinecompounds of this invention includes asymmetric carbon atoms. It is tobe understood accordingly that the isomers arising from such asymmetry(e.g., all enantiomers and diastereomers) are included within the scopeof this invention, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis. Furthermore, thestructures and other compounds and moieties discussed in thisapplication also include all tautomers thereof.

7. Methods for Treating Tetracycline Responsive States

The invention also pertains to methods for treating a tetracyclineresponsive states in subjects, by administering to a subject aneffective amount of a tetracycline compound of the invention (e.g., acompound of Formula I, II, III, IV, V or otherwise described herein),such that the tetracycline responsive state is treated.

The term “treating” includes curing as well as ameliorating at least onesymptom of the state, disease or disorder, e.g., the tetracyclinecompound responsive state.

The language “tetracycline compound responsive state” or “tetracyclineresponsive state” includes states which can be treated, prevented, orotherwise ameliorated by the administration of a tetracycline compoundof the invention, e.g., a 3, 10, and/or 12a substituted tetracyclinecompound. Tetracycline compound responsive states include bacterial,viral, and fungal infections (including those which are resistant toother tetracycline compounds), cancer (e.g., prostate, breast, colon,lung melanoma and lymph cancers and other disorders characterized byunwanted cellular proliferation, including, but not limited to, thosedescribed in U.S. Pat. No. 6,100,248), arthritis, osteoporosis,diabetes, and other states for which tetracycline compounds have beenfound to be active (see, for example, U.S. Pat. Nos. 5,789,395;5,834,450; 6,277,061 and 5,532,227, each of which is expresslyincorporated herein by reference). Compounds of the invention can beused to prevent or control important mammalian and veterinary diseasessuch as diarrhea, urinary tract infections, infections of skin and skinstructure, ear, nose and throat infections, wound infection, mastitisand the like. In addition, methods for treating neoplasms usingtetracycline compounds of the invention are also included (van derBozert et al., Cancer Res., 48:6686-6690 (1988)). In a furtherembodiment, the tetracycline responsive state is not a bacterialinfection. In another embodiment, the tetracycline compounds of theinvention are essentially non-antibacterial. For example,non-antibacterial tetracycline compounds of the invention may have MICvalues greater than about 4 μg/ml (as measured by assays known in theart and/or the assay given in Example 2).

Tetracycline compound responsive states also include inflammatoryprocess associated states (IPAS). The term “inflammatory processassociated state” includes states in which inflammation or inflammatoryfactors (e.g., matrix metalloproteinases (MMPs), nitric oxide (NO), TNF,interleukins, plasma proteins, cellular defense systems, cytokines,lipid metabolites, proteases, toxic radicals, adhesion molecules, etc.)are involved or are present in an area in aberrant amounts, e.g., inamounts which may be advantageous to alter, e.g., to benefit thesubject. The inflammatory process is the response of living tissue todamage. The cause of inflammation may be due to physical damage,chemical substances, micro-organisms, tissue necrosis, cancer or otheragents. Acute inflammation is short-lasting, lasting only a few days. Ifit is longer lasting however, then it may be referred to as chronicinflammation.

IPAF's include inflammatory disorders. Inflammatory disorders aregenerally characterized by heat, redness, swelling, pain and loss offunction. Examples of causes of inflammatory disorders include, but arenot limited to, microbial infections (e.g., bacterial and fungalinfections), physical agents (e.g., burns, radiation, and trauma),chemical agents (e.g., toxins and caustic substances), tissue necrosisand various types of immunologic reactions.

Examples of inflammatory disorders include, but are not limited to,osteoarthritis, rheumatoid arthritis, acute and chronic infections(bacterial and fungal, including diphtheria and pertussis); acute andchronic bronchitis, sinusitis, and upper respiratory infections,including the common cold; acute and chronic gastroenteritis andcolitis; acute and chronic cystitis and urethritis; acute and chronicdermatitis; acute and chronic conjunctivitis; acute and chronicserositis (pericarditis, peritonitis, synovitis, pleuritis andtendinitis); uremic pericarditis; acute and chronic cholecystis; acuteand chronic vaginitis; acute and chronic uveitis; drug reactions; insectbites; burns (thermal, chemical, and electrical); and sunburn.

Tetracycline compound responsive states also include NO associatedstates. The term “NO associated state” includes states which involve orare associated with nitric oxide (NO) or inducible nitric oxide synthase(iNOS). NO associated state includes states which are characterized byaberrant amounts of NO and/or iNOS. Preferably, the NO associated statecan be treated by administering tetracycline compounds of the invention,e.g., a 3, 10, and/or 12a substituted tetracycline compound. Thedisorders, diseases and states described in U.S. Pat. Nos. 6,231,894;6,015,804; 5,919,774; and 5,789,395 are also included as NO associatedstates. The entire contents of each of these patents are herebyincorporated herein by reference.

Other examples of NO associated states include, but are not limited to,malaria, senescence, diabetes, vascular stroke, neurodegenerativedisorders (Alzheimer's disease & Huntington's disease), cardiac disease(reperfusion-associated injury following infarction), juvenile diabetes,inflammatory disorders, osteoarthritis, rheumatoid arthritis, acute,recurrent and chronic infections (bacterial, viral and fungal); acuteand chronic bronchitis, sinusitis, and respiratory infections, includingthe common cold; acute and chronic gastroenteritis and colitis; acuteand chronic cystitis and urethritis; acute and chronic dermatitis; acuteand chronic conjunctivitis; acute and chronic serositis (pericarditis,peritonitis, synovitis, pleuritis and tendonitis); uremic pericarditis;acute and chronic cholecystis; cystic fibrosis, acute and chronicvaginitis; acute and chronic uveitis; drug reactions; insect bites;burns (thermal, chemical, and electrical); and sunburn.

The term “inflammatory process associated state” also includes, in oneembodiment, matrix metalloproteinase associated states (MMPAS). MMPASinclude states charachterized by abberrant amounts of MMPs or MMPactivity. These are also include as tetracycline compound responsivestates which may be treated using compounds of the invention, e.g., 3,10, and/or 12a substituted tetracycline compounds.

Examples of matrix metalloproteinase associated states (“MMPAS's”)include, but are not limited to, arteriosclerosis, corneal ulceration,emphysema, osteoarthritis, multiple sclerosis (Liedtke et al., Ann.Neurol. 1998, 44:35-46; Chandler et al., J. Neuroimmunol. 1997,72:155-71), osteosarcoma, osteomyelitis, bronchiectasis, chronicpulmonary obstructive disease, skin and eye diseases, periodontitis,osteoporosis, rheumatoid arthritis, ulcerative colitis, inflammatorydisorders, tumor growth and invasion (Stetler-Stevenson et al., Annu.Rev. Cell Biol. 1993, 9:541-73; Tryggvason et al., Biochim. Biophys.Acta 1987, 907:191-217; Li et al., Mol. Carcinog. 1998, 22:84-89)),metastasis, acute lung injury, stroke, ischemia, diabetes, aortic orvascular aneurysms, skin tissue wounds, dry eye, bone and cartilagedegradation (Greenwald et al., Bone 1998, 22:33-38; Ryan et al., Curr.Op. Rheumatol. 1996, 8; 238-247). Other MMPAS include those described inU.S. Pat. Nos. 5,459,135; 5,321,017; 5,308,839; 5,258,371; 4,935,412;4,704,383, 4,666,897, and RE 34,656, incorporated herein by reference intheir entirety.

In another embodiment, the tetracycline compound responsive state iscancer. Examples of cancers which the tetracycline compounds of theinvention may be useful to treat include all solid tumors, i.e.,carcinomas e.g., adenocarcinomas, and sarcomas. Adenocarcinomas arecarcinomas derived from glandular tissue or in which the tumor cellsform recognizable glandular structures. Sarcomas broadly include tumorswhose cells are embedded in a fibrillar or homogeneous substance likeembryonic connective tissue. Examples of carcinomas which may be treatedusing the methods of the invention include, but are not limited to,carcinomas of the prostate, breast, ovary, testis, lung, colon, andbreast. The methods of the invention are not limited to the treatment ofthese tumor types, but extend to any solid tumor derived from any organsystem. Examples of treatable cancers include, but are not limited to,colon cancer, bladder cancer, breast cancer, melanoma, ovariancarcinoma, prostatic carcinoma, lung cancer, and a variety of othercancers as well. The methods of the invention also cause the inhibitionof cancer growth in adenocarcinomas, such as, for example, those of theprostate, breast, kidney, ovary, testes, and colon.

In an embodiment, the tetracycline responsive state of the invention iscancer. The invention pertains to a method for treating a subjectsuffering or at risk of suffering from cancer, by administering aneffective amount of a substituted tetracycline compound, such thatinhibition cancer cell growth occurs, i.e., cellular proliferation,invasiveness, metastasis, or tumor incidence is decreased, slowed, orstopped. The inhibition may result from inhibition of an inflammatoryprocess, down-regulation of an inflammatory process, some othermechanism, or a combination of mechanisms. Alternatively, thetetracycline compounds may be useful for preventing cancer recurrence,for example, to treat residual cancer following surgical resection orradiation therapy. The tetracycline compounds useful according to theinvention are especially advantageous as they are substantiallynon-toxic compared to other cancer treatments. In a further embodiment,the compounds of the invention are administered in combination withstandard cancer therapy, such as, but not limited to, chemotherapy.

Examples of tetracycline responsive states also include neurologicaldisorders which include both neuropsychiatric and neurodegenerativedisorders, but are not limited to, such as Alzheimer's disease,dementias related to Alzheimer's disease (such as Pick's disease),Parkinson's and other Lewy diffuse body diseases, senile dementia,Huntington's disease, Gilles de la Tourette's syndrome, multiplesclerosis, amylotrophic lateral sclerosis (ALS), progressivesupranuclear palsy, epilepsy, and Creutzfeldt-Jakob disease; autonomicfunction disorders such as hypertension and sleep disorders, andneuropsychiatric disorders, such as depression, schizophrenia,schizoaffective disorder, Korsakoff's psychosis, mania, anxietydisorders, or phobic disorders; learning or memory disorders, e.g.,amnesia or age-related memory loss, attention deficit disorder,dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), bipolar affectiveneurological disorders, e.g., migraine and obesity. Further neurologicaldisorders include, for example, those listed in the American PsychiatricAssociation's Diagnostic and Statistical manual of Mental Disorders(DSM), the most current version of which is incorporated herein byreference in its entirety.

Other examples of tetracycline compound responsive states are describedin WO 03/005971A2, U.S. Ser. No. 60/421,248, and U.S. Ser. No.60/480,482, each incorporated herein by reference.

The language “in combination with” another therapeutic agent ortreatment includes co-administration of the tetracycline compound,(e.g., inhibitor) and with the other therapeutic agent or treatment,administration of the tetracycline compound first, followed by the othertherapeutic agent or treatment and administration of the othertherapeutic agent or treatment first, followed by the tetracyclinecompound. The other therapeutic agent may be any agent which is known inthe art to treat, prevent, or reduce the symptoms of an IPAS.Furthermore, the other therapeutic agent may be any agent of benefit tothe patient when administered in combination with the administration ofan tetracycline compound. In one embodiment, the cancers treated bymethods of the invention include those described in U.S. Pat. Nos.6,100,248; 5,843,925; 5,837,696; or 5,668,122, incorporated herein byreference in their entirety.

In another embodiment, the tetracycline compound responsive state isdiabetes, e.g., juvenile diabetes, diabetes mellitus, diabetes type I,or diabetes type II. In a further embodiment, protein glycosylation isnot affected by the administration of the tetracycline compounds of theinvention. In another embodiment, the tetracycline compound of theinvention is administered in combination with standard diabetictherapies, such as, but not limited to insulin therapy. In a furtherembodiment, the IPAS includes disorders described in U.S. Pat. Nos.5,929,055; and 5,532,227, incorporated herein by reference in theirentirety.

In another embodiment, the tetracycline compound responsive state is abone mass disorder. Bone mass disorders include disorders where asubjects bones are disorders and states where the formation, repair orremodeling of bone is advantageous. For examples bone mass disordersinclude osteoporosis (e.g., a decrease in bone strength and density),bone fractures, bone formation associated with surgical procedures(e.g., facial reconstruction), osteogenesis imperfecta (brittle bonedisease), hypophosphatasia, Paget's disease, fibrous dysplasia,osteopetrosis, myeloma bone disease, and the depletion of calcium inbone, such as that which is related to primary hyperparathyroidism. Bonemass disorders include all states in which the formation, repair orremodeling of bone is advantageous to the subject as well as all otherdisorders associated with the bones or skeletal system of a subjectwhich can be treated with the tetracycline compounds of the invention.In a further embodiment, the bone mass disorders include those describedin U.S. Pat. Nos. 5,459,135; 5,231,017; 5,998,390; 5,770,588; RE 34,656;5,308,839; 4,925,833; 3,304,227; and 4,666,897, each of which is herebyincorporated herein by reference in its entirety.

In another embodiment, the tetracycline compound responsive state isacute lung injury. Acute lung injuries include adult respiratorydistress syndrome (ARDS), post-pump syndrome (PPS), and trauma. Traumaincludes any injury to living tissue caused by an extrinsic agent orevent. Examples of trauma include, but are not limited to, crushinjuries, contact with a hard surface, or cutting or other damage to thelungs.

The invention also pertains to a method for treating acute lung injuryby administering a substituted tetracycline compound of the invention.

The tetracycline responsive states of the invention also include chroniclung disorders. The invention pertains to methods for treating chroniclung disorders by administering a tetracycline compound, such as thosedescribed herein. The method includes administering to a subject aneffective amount of a substituted tetracycline compound such that thechronic lung disorder is treated. Examples of chronic lung disordersinclude, but are not limited, to asthma, cystic fibrosis, and emphysema.In a further embodiment, the tetracycline compounds of the inventionused to treat acute and/or chronic lung disorders such as thosedescribed in U.S. Pat. Nos. 5,977,091; 6,043,231; 5,523,297; and5,773,430, each of which is hereby incorporated herein by reference inits entirety.

In yet another embodiment, the tetracycline compound responsive state isischemia, stroke, or ischemic stroke. The invention also pertains to amethod for treating ischemia, stroke, or ischemic stroke byadministering an effective amount of a substituted tetracycline compoundof the invention. In a further embodiment, the tetracycline compounds ofthe invention are used to treat such disorders as described in U.S. Pat.Nos. 6,231,894; 5,773,430; 5,919,775 or 5,789,395, incorporated hereinby reference.

In another embodiment, the tetracycline compound responsive state is askin wound. The invention also pertains, at least in part, to a methodfor improving the healing response of the epithelialized tissue (e.g.,skin, mucusae) to acute traumatic injury (e.g., cut, burn, scrape,etc.). The method may include using a tetracycline compound of theinvention (which may or may not have antibacterial activity) to improvethe capacity of the epithelialized tissue to heal acute wounds. Themethod may increase the rate of collagen accumulation of the healingtissue. The method may also decrease the proteolytic activity in theepthithelialized tissue by decreasing the collagenolytic and/orgellatinolytic activity of MMPs. In a further embodiment, thetetracycline compound of the invention is administered to the surface ofthe skin (e.g., topically). In a further embodiment, the tetracyclinecompound of the invention used to treat a skin wound, and other suchdisorders as described in, for example, U.S. Pat. Nos. 5,827,840;4,704,383; 4,935,412; 5,258,371; 5,308,8391 5,459,135; 5,532,227; and6,015,804; each of which is incorporated herein by reference in itsentirety.

In yet another embodiment, the tetracycline compound responsive state isan aortic or vascular aneurysm in vascular tissue of a subject (e.g., asubject having or at risk of having an aortic or vascular aneurysm,etc.). The tetracycline compound may by effective to reduce the size ofthe vascular aneurysm or it may be administered to the subject prior tothe onset of the vascular aneurysm such that the aneurysm is prevented.In one embodiment, the vascular tissue is an artery, e.g., the aorta,e.g., the abdominal aorta. In a further embodiment, the tetracyclinecompounds of the invention are used to treat disorders described in U.S.Pat. Nos. 6,043,225 and 5,834,449, incorporated herein by reference intheir entirety.

Bacterial infections may be caused by a wide variety of gram positiveand gram negative bacteria. The compounds of the invention are useful asantibiotics against organisms which are resistant to other tetracyclinecompounds. The antibiotic activity of the tetracycline compounds of theinvention may be determined using the method discussed in Example 2, orby using the in vitro standard broth dilution method described in Waitz,J. A., National Commission for Clinical Laboratory Standards, DocumentM7-A2, vol. 10, no. 8, pp. 13-20, 2^(nd) edition, Villanova, Pa. (1990).

The tetracycline compounds may also be used to treat infectionstraditionally treated with tetracycline compounds such as, for example,rickettsiae; a number of gram-positive and gram-negative bacteria; andthe agents responsible for lymphogranuloma venereum, inclusionconjunctivitis, psittacosis. The tetracycline compounds may be used totreat infections of, e.g., K. pneumoniae, Salmonella, E. hirae, A.baumanii, B. catarrhalis, H. influenzae, P. aeruginosa, E. faecium, E.coli, S. aureus or E. faecalis. In one embodiment, the tetracyclinecompound is used to treat a bacterial infection that is resistant toother tetracycline antibiotic compounds. The tetracycline compound ofthe invention may be administered with a pharmaceutically acceptablecarrier.

The language “effective amount” of the compound is that amount necessaryor sufficient to treat or prevent a tetracycline compound responsivestate. The effective amount can vary depending on such factors as thesize and weight of the subject, the type of illness, or the particulartetracycline compound. For example, the choice of the tetracyclinecompound can affect what constitutes an “effective amount”. One ofordinary skill in the art would be able to study the aforementionedfactors and make the determination regarding the effective amount of thetetracycline compound without undue experimentation.

The invention also pertains to methods of treatment againstmicroorganism infections and associated diseases. The methods includeadministration of an effective amount of one or more tetracyclinecompounds to a subject. The subject can be either a 3′5 plant or,advantageously, an animal, e.g., a mammal, e.g., a human.

In the therapeutic methods of the invention, one or more tetracyclinecompounds of the invention may be administered alone to a subject, ormore typically a compound of the invention will be administered as partof a pharmaceutical composition in mixture with conventional excipient,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for parenteral, oral or other desired administrationand which do not deleteriously react with the active compounds and arenot deleterious to the recipient thereof.

7. Pharmaceutical Compositions of the Invention

The invention also pertains to pharmaceutical compositions comprising atherapeutically effective amount of a tetracycline compound (e.g., acompound of Formula I, II, III, IV, V or any other compound describedherein) and, optionally, a pharmaceutically acceptable carrier.

The language “pharmaceutically acceptable carrier” includes substancescapable of being coadministered with the tetracycline compound(s), andwhich allow both to perform their intended function, e.g., treat orprevent a tetracycline responsive state. Suitable pharmaceuticallyacceptable carriers include but are not limited to water, saltsolutions, alcohol, vegetable oils, polyethylene glycols, gelatin,lactose, amylose, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds of the invention.

The tetracycline compounds of the invention that are basic in nature arecapable of forming a wide variety of salts with various inorganic andorganic acids. The acids that may be used to prepare pharmaceuticallyacceptable acid addition salts of the tetracycline compounds of theinvention that are basic in nature are those that form non-toxic acidaddition salts, i.e., salts containing pharmaceutically acceptableanions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate,sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate,lactate, salicylate, citrate, acid citrate, tartrate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand palmoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts.Although such salts must be pharmaceutically acceptable foradministration to a subject, e.g., a mammal, it is often desirable inpractice to initially isolate a tetracycline compound of the inventionfrom the reaction mixture as a pharmaceutically unacceptable salt andthen simply convert the latter back to the free base compound bytreatment with an alkaline reagent and subsequently convert the latterfree base to a pharmaceutically acceptable acid addition salt. The acidaddition salts of the base compounds of this invention are readilyprepared by treating the base compound with a substantially equivalentamount of the chosen mineral or organic acid in an aqueous solventmedium or in a suitable organic solvent, such as methanol or ethanol.Upon careful evaporation of the solvent, the desired solid salt isreadily obtained. The preparation of other tetracycline compounds of theinvention not specifically described in the foregoing experimentalsection can be accomplished using combinations of the reactionsdescribed above that will be apparent to those skilled in the art.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention that are acidic in natureare capable of forming a wide variety of base salts. The chemical basesthat may be used as reagents to prepare pharmaceutically acceptable basesalts of those tetracycline compounds of the invention that are acidicin nature are those that form non-toxic base salts with such compounds.Such non-toxic base salts include, but are not limited to those derivedfrom such pharmaceutically acceptable cations such as alkali metalcations (e.g., potassium and sodium) and alkaline earth metal cations(e.g., calcium and magnesium), ammonium or water-soluble amine additionsalts such as N-methylglucamine-(meglumine), and the loweralkanolammonium and other base salts of pharmaceutically acceptableorganic amines. The pharmaceutically acceptable base addition salts oftetracycline compounds of the invention that are acidic in nature may beformed with pharmaceutically acceptable cations by conventional methods.Thus, these salts may be readily prepared by treating the tetracyclinecompound of the invention with an aqueous solution of the desiredpharmaceutically acceptable cation and evaporating the resultingsolution to dryness, preferably under reduced pressure. Alternatively, alower alkyl alcohol solution of the tetracycline compound of theinvention may be mixed with an alkoxide of the desired metal and thesolution subsequently evaporated to dryness.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention and pharmaceuticallyacceptable salts thereof can be administered via either the oral,parenteral or topical routes. In general, these compounds are mostdesirably administered in effective dosages, depending upon the weightand condition of the subject being treated and the particular route ofadministration chosen. Variations may occur depending upon the speciesof the subject being treated and its individual response to saidmedicament, as well as on the type of pharmaceutical formulation chosenand the time period and interval at which such administration is carriedout.

The pharmaceutical compositions of the invention may be administeredalone or in combination with other known compositions for treatingtetracycline responsive states in a subject, e.g., a mammal. Preferredmammals include pets (e.g., cats, dogs, ferrets, etc.), farm animals(cows, sheep, pigs, horses, goats, etc.), lab animals (rats, mice,monkeys, etc.), and primates (chimpanzees, humans, gorillas). Thelanguage “in combination with” a known composition is intended toinclude simultaneous administration of the composition of the inventionand the known composition, administration of the composition of theinvention first, followed by the known composition and administration ofthe known composition first, followed by the composition of theinvention. Any of the therapeutically composition known in the art fortreating tetracycline responsive states can be used in the methods ofthe invention.

The tetracycline compounds of the invention may be administered alone orin combination with pharmaceutically acceptable carriers or diluents byany of the routes previously mentioned, and the administration may becarried out in single or multiple doses. For example, the noveltherapeutic agents of this invention can be administered advantageouslyin a wide variety of different dosage forms, i.e., they may be combinedwith various pharmaceutically acceptable inert carriers in the form oftablets, capsules, lozenges, troches, hard candies, powders, sprays(e.g., aerosols, etc.), creams, salves, suppositories, jellies, gels,pastes, lotions, ointments, aqueous suspensions, injectable solutions,elixirs, syrups, and the like. Such carriers include solid diluents orfillers, sterile aqueous media and various non-toxic organic solvents,etc. Moreover, oral pharmaceutical compositions can be suitablysweetened and/or flavored. In general, the therapeutically-effectivecompounds of this invention are present in such dosage forms atconcentration levels ranging from about 5.0% to about 70% by weight.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (and preferably corn, potato or tapioca starch), alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active ingredient may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof. The compositions of the invention may beformulated such that the tetracycline compositions are released over aperiod of time after administration.

For parenteral administration (including intraperitoneal, subcutaneous,intravenous, intradermal or intramuscular injection), solutions of atherapeutic compound of the present invention in either sesame or peanutoil or in aqueous propylene glycol may be employed. The aqueoussolutions should be suitably buffered (preferably pH greater than 8) ifnecessary and the liquid diluent first rendered isotonic. These aqueoussolutions are suitable for intravenous injection purposes. The oilysolutions are suitable for intraarticular, intramuscular andsubcutaneous injection purposes. The preparation of all these solutionsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well known to those skilled in the art. Forparenteral application, examples of suitable preparations includesolutions, preferably oily or aqueous solutions as well as suspensions,emulsions, or implants, including suppositories. Therapeutic compoundsmay be formulated in sterile form in multiple or single dose formatssuch as being dispersed in a fluid carrier such as sterile physiologicalsaline or 5% saline dextrose solutions commonly used with injectables.

Additionally, it is also possible to administer the compounds of thepresent invention topically when treating inflammatory conditions of theskin. Examples of methods of topical administration include transdermal,buccal or sublingual application. For topical applications, therapeuticcompounds can be suitably admixed in a pharmacologically inert topicalcarrier such as a gel, an ointment, a lotion or a cream. Such topicalcarriers include water, glycerol, alcohol, propylene glycol, fattyalcohols, triglycerides, fatty acid esters, or mineral oils. Otherpossible topical carriers are liquid petrolatum, isopropylpalmitate,polyethylene glycol, ethanol 95%, polyoxyethylene monolauriate 5% inwater, sodium lauryl sulfate 5% in water, and the like. In addition,materials such as anti-oxidants, humectants, viscosity stabilizers andthe like also may be added if desired.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or carbohydrate carrier binder or the like, thecarrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

In addition to treatment of human subjects, the therapeutic methods ofthe invention also will have significant veterinary applications, e.g.for treatment of livestock such as cattle, sheep, goats, cows, swine andthe like; poultry such as chickens, ducks, geese, turkeys and the like;horses; and pets such as dogs and cats. Also, the compounds of theinvention may be used to treat non-animal subjects, such as plants.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, the particular site of administration, etc. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines.

In general, compounds of the invention for treatment can be administeredto a subject in dosages used in prior tetracycline therapies. See, forexample, the Physicians' Desk Reference. For example, a suitableeffective dose of one or more compounds of the invention will be in therange of from 0.01 to 100 milligrams per kilogram of body weight ofrecipient per day, preferably in the range of from 0.1 to 50 milligramsper kilogram body weight of recipient per day, more preferably in therange of 1 to 20 milligrams per kilogram body weight of recipient perday. The desired dose is suitably administered once daily, or severalsub-doses, e.g. 2 to 5 sub-doses, are administered at appropriateintervals through the day, or other appropriate schedule.

It will also be understood that normal, conventionally known precautionswill be taken regarding the administration of tetracyclines generally toensure their efficacy under normal use circumstances. Especially whenemployed for therapeutic treatment of humans and animals in vivo, thepractitioner should take all sensible precautions to avoidconventionally known contradictions and toxic effects. Thus, theconventionally recognized adverse reactions of gastrointestinal distressand inflammations, the renal toxicity, hypersensitivity reactions,changes in blood, and impairment of absorption through aluminum,calcium, and magnesium ions should be duly considered in theconventional manner.

Furthermore, the invention also pertains to the use of a tetracyclinecompound of formula I, II, III, IV, V, or any other compound describedherein, for the preparation of a medicament. The medicament may includea pharmaceutically acceptable carrier and the tetracycline compound isan effective amount, e.g., an effective amount to treat a tetracyclineresponsive state.

EXEMPLIFICATION OF THE INVENTION Example 1: Synthesis of SelectedCompounds of the Invention

The above compound was prepared from 7-iodo-sancycline (15.0 g, 22.9mmol) combined with Pd(dppf)₂Cl₂ (1.7 g, 2.29 mmol) and DMF (300 mL) ina 1 L round bottom 2 neck flask. Na₂CO₃ (7.2 g, 68.2 mmol) was dissolvedin water (15 mL) was added to reaction solution.2-fluoro-pyridine-5-boronic acid (6.4 g, 45.9 mmol) was dissolved in DMF(25 mL) and also added to reaction solution. Reaction mixture wasstirred at 65° C. (oil bath temperature) under an argon atmosphere andreaction was monitored by HPLC and LC/MS. Reaction shown to be completewithin 3 hr. Filtered through celite and evaporated solvent in vacuo.Redissolved in MeOH (30 mL) and precipitated in MTBE (3 L) to produce ayellow precipitate. Filtered and dried under vacuum overnight to yield15 g of yellow powder. This crude material (9 g, 17.8 mmol) wasdissolved in TFA/Triflic acid (83 mL/7 mL) and cooled to 0° C. using anice bath. N-iodo-succinimide (8 g, 35.6 mmol) was added portionwise toreaction solution over 2 hr. Reaction complete after 3 hrs—and 20% moreNIS added to reaction. Evaporated TFA in vacuo and precipitatedremaining acid in MTBE (1.4 L) at room temp. Yellow precipitate.Filtered and dried under vacuum overnight to yield 8.4 g of crudeproduct. This crude material (4 g, 6.3 mmol) was combined with NaOAc(0.52 g, 6.3 mmol) in an oven-dried 250 mL 2 neck round bottom flask.Anhydrous DMF (60 mL) was syringed into reaction flask. Stirred underargon at room temp 1 hr. Diluted with more anhydrous DMF (120 mL) and aCO-filled balloon was placed on top neck of reaction flask. CO waspurged through reaction direction from lecture bottle for 15 min. Flaskthen open to CO-filled balloon and allowed to stir at 60° C. (oil bathtemp) while Pd(PPh₃)₄ (2.2 g, 1.9 mmol) was added as a DMF slurry viasyringe. Stirred at temperature 1 hr. SnBu₃H (1.6 g, 6.3 mmol) was addedvia syringe pump over 2 hr. Reaction monitored by HPLC and LC/MS andshown to be complete upon completion of tin addition. Evaporated solventin vacuo. Purified by preparative HPLC in 20% yield in preparation forfinal synthesis step. This purified material (0.25 g, 0.46 mmol) wascombined with anhydrous DMF (15 mL) in an oven-dried 100 mL flask. InCl₃(0.005 g, 0.023 mmol), N-methyl-allylamine (0.17 g, 0.23 mmol) wereadded to reaction and stirred at room temperature under argon 1 hr.NaCNBH₃ (0.035 g, 0.55 mmol) was added to reaction solution and wasmonitored by HPLC and LC/NIS. Reaction 80% complete within 6 hrs ofreaction time. Evaporated solvent in vacuo. Final product was isolatedby preparative HPLC in 10% yield as a yellow solid. ESI-MS: m/z (M+H)593.

7-Ethyl-9-(4′,4′-Difluoro-N-Piperidinyl methyl)-Sancycline

The compound was prepared from 7-ethyl-9-formyl-sancycline (0.23 g, 0.49mmol) combined with InCl₃ (0.011 g, 0.049 mmol),4,4-difluoropiperidine.HCl (0.17 g, 0.98 mmol), Et₃N (0.099 g, 0.98mmol), and DMF (8 mL) in a glass vial. Stirred under argon at roomtemperature 30 min. NaCNBH₃ (0.043 g, 0.69 mmol) was added to reactionvial and continued to stir at room temperature under argon. Reaction wasmonitored by LC/MS and HPLC and shown to be complete in 2 hrs. Quenchedreaction with MeOH (15 mL) and evaporated solvent in vacuo. Product wasisolated by preparative HPLC in 20% yield as a yellow solid. ESI-MS: m/z(M+H) 576.

7-(Trifluoroalkenyl)-9-(2′-trans-2-methyl-2-butene) aminomethylSancycline

To a stirred solution of powered Zn (5.00 g, 76.5 mmol) in dry THF (50.0mL) at 0 C was added iodo-trifluor alkene (2.00 mL, 4.50 g, 21.0 mmol)slowly over a 0.5 h time period. The reaction was stirred for anadditional 1.5 h before it was filtered under an inert atmosphere andreduced of all solvent using rotary evaporation (25.0 C, 5.00 mm Hg) toyield the trifluoro-zinc-iodo-alkene reagent (approximately 3 mL). DryDMF (10 mL) was added to the above zinc-reagent and this solution wasadded to a stirred solution of 7-Iodo-9-trans-2-methyl-2-butenesancycline free base (1.00 g, 1.57 mmol) andtetrakis(triphenylphosphine)palladium (0.181 g, 0.156 mmol) in dry DMF(10 mL). The contents were heated to 40 C and allowed to stir for 20minutes. The reaction was then filtered and purified using reverse phaseHPLC to give 7-trifluoroalkene sancycline product (557 mg, 0.0942 mmol,60% yield) LCMS m/z=592.2392 (M+H).

7-(2′-Pyrazinyl)-9-(3′,3′,3′-Trifluoro-propylamino)-methyl-Sancycline

Step 1:

7-Iodo-9-aminomethyl sancycline (569 mg, 1 mmol), indium trichloride (22mg, 0.1 mmol) and trifluoropropionaldehyde (224 μL, 2 mmol) were takenin DMF (25 mL) and stirred at room temperature for 10 minutes. To thissolution, sodium triacetoxyborohydride (635 mg, 3 mmol) was added atonce and the reaction mixture was stirred at room temperature foranother 30 minutes. Progress of the reaction was monitored by HPLC andLC/MS. Reaction was completed in 30 minutes. DMF was then removed andthe crude material obtained was then precipitated using diethylether/MeOH (100/10 mL). Filteration of the precipitate gave a yellowpowder, which was used for the next step without further purification.

Step 2:

7-Iodo-9-(3,3,3-trifluoro-propylamino)-methyl-sancycline (665 mg, 1mmol), Pd(PPh₃)₄ (115 mg, 0.1 mmol), Pd(OAc)₂ (22 mg, 0.1 mmol), CuI (19mg, 0.1 mmol) were taken in anhydrous DMF (30 mL) and purged with argonfor 5 minutes. To this solution, 2-pyrazine-stannane (738 mg, 2 mmol)was added and the reaction mixture was stirred at room temperature for 2hours. Reaction was completed by then (monitored by HPLC/LCMS). It wasthen filtered through celite, washed with 5 mL of methanol. Solvent wasevaporated to dryness. The crude material obtained was purified usingpreparative HPLC. A yellow solid was obtained after evaporating thefractions, which was converted to its HCl salt using MeOH/HCl solution.LC-MS (M+1 618).

7-Amino-9-Iodo-Doxycycline

To 500 mg of 9-iodo-doxycycline in 10 ml of methanesulfonic acid wasadded 1.1 eq. of sodium nitrate. The reaction mixture was left stirringfor several hrs and was monitored by analytical HPLC. The intermediate(9-Iodo-7-nitro-doxycycline) was isolated by diluting he solution withice-water, adjusting the pH with sodium hydroxide (pH ˜4) and extractingthe product with n-butanol. The solvent was evaporated under reducedpressure and the crude material was subjected to hydrogenation using 10%Pd/C in methanol. The final product was obtained via preparative HPLC.The LCMS showed the desired material; MS: 586. The structure wasconfirmed by NMR.

7-(Dimethylamino)-9-(4′,4′-Difluoropiperdinyl)-Doxycycline

To a solution of 105 mg (0.16 mmol) of9-(4-difluoropiperdinyl)-doxycycline dihydrochloride in 10 mL ofmethanesulfonic acid at room temperature, was added 19.4 mg (0.19 mmol)of potassium nitrate dissolved in 4 mL of methanesulfonic acid. Thereaction was monitored by LCMS. After 30 minutes, the reaction mixturewas poured over ice and diluted to 160 mL with ice water. The solutionwas loaded onto a 2.5×1 cm column of divinylbenzene resin (1000angstrom, 5-25 μm) equilibrated with water. The crude reaction mixturewas washed with excess water to remove methanesulfonic acid followed anexcess of 1N ammonium acetate to neutralize the crude mixture. Theexcess ammonium acetate was removed by a water wash and the crudecompound was purified by elution with 40% methanol in water with 0.1%HCl. The purified material was evaporated to dryness to yield 70 mg of9-(4-difluoropiperdinyl)-7-nitro-doxycycline as the dihydrochloride salt(Yield=63%). LCMS (MH+) 623. To 70 mg (0.10 mmol) of9-(4-difluoropiperdinyl)-7-nitro-doxycycline dihydrochloride in 20 mL ofmethoxyethanol was added 200 mL of sulfuric acid and 162 mL (2 mmol) of37% formaldehyde in water. The reaction mixture was purged with Argongas and 40 mg of 10% wet Palladium on carbon was added with stirring.The reaction was hydrogenated at room temperature and 760 torr hydrogengas for 12 hours. The crude reaction was passed through Celite andevaporated to dryness. The crude reaction mixture was purified bypreparative HPLC (1 inch×25 cm, Phenomenex Luna C18, 10 mm, Gradient5-40% B buffer, A=water+0.1% TFA, B=acetonitrile+0.1% TFA, detection at280 nm) to yield 20 mg of the product as the dihydrochloride salt(Yield=30%). LCMS (MH+) 621.

7-Diethylamino-9-(4′-Fluoro-N-Piperidinyl methyl)-Sancycline

7-NH₂-sancycline (4.0 g, 9.32 mmol) was combined with 2-methoxyethanol(100 mL), H₂SO₄ (5 mL of 1N solution) in a 2-neck 250 mL round bottomflask. Acetaldehyde (5.2 mL, 9.32 mmol) was added to reaction solutionand contents were stirred at room temperature under argon for 20minutes. Pd/C (1.25 g) was added to reaction and contents wereevacuated/flushed with argon 3 times. A balloon filled with H₂ wasplaced on top neck of reaction flask and reaction solution wasevacuated/flushed with H₂ three times. The reaction was stirredovernight under H₂ pressure at room temperature. The reaction wasmonitored by HPLC and LC/MS and shown to be complete by morning. Themixture was filtered through celite and solvent evaporated in vacuo. Theresidue was redissolved in water (1 L) and the pH was adjusted with Et₃Nto pH-5. The mixture was filtered again through celite and loaded onto aDVB column. The compound eluted at 15% CH₃CN. Clean fractions wereevaporated and dried overnight under vacuum. A yellow/brown solid(7-diethylamino sancycline) was isolated in 40% yield.

7-diethylamino sancycline (1.4 g, 2.88 mmol) was dissolved inTFA/Triflic acid (22 mL/6 mL) in a 100 mL flask. N-iodosuccinimide (1.2g, 5.78 mmol) was added portionwise to reaction solution every 20minutes. The reaction monitored by HPLC and LC/MS and shown to becomplete within 3 hours. The reaction solution was diluted with H₂O(0.1% TFA) (30 mL) and the solvent was evaporated. The residue wasredissolved in H₂O (100 mL) and loaded onto a 5 g DVB cartridge. Thecrude product eluted at 30-50% CH₃CN. A yellow/brown crude product wasisolated in 90% yield.

This crude material, 7-diethylamino-9-iodo-sancycline, (1.8 g, 2.95mmol) was dissolved in anhydrous DMF (100 mL) in a 2 neck 1 L roundbottom flask and placed under argon. NaOAc (0.61 g, 7.36 mmol) was addedto reaction solution and stirred at room temperature 45 min. Pd(PPh₃)₄(1.02 g, 8.85 mmol) was added to reaction and a CO-filled balloon wasplaced on top neck of reaction flask. CO was bubbled through reactionsolution for 10 min. then flask opened to CO balloon. SnBu₃H (0.8 g,2.95 mmol) was added via syringe pump to reaction solution over 1 hourwhile heating to 65° C. (oil bath temperature). The reaction wasmonitored by LC/MS and shown to be complete upon addition of tinhydride. H₂O (0.1% TFA, 0.3 L) was added to the reaction flask and aprecipitate formed. The mixture was filtered through celite and thefiltrate was evaporated in vacuo. A brown solid in 90% yield (crudematerial) was isolated.

7-diethylamino-9-formyl-sancycline (0.25 g, 0.49 mmol) was dissolved inDMF (10 mL). InCl₃ (0.01 g, 0.049 mmol), 4-fluoropiperidine.HCl (0.15 g,0.98 mmol), and Et₃N (0.09 g, 0.98 mmol) were added to reactionsolution. The reaction was stirred at room temperature under argon 45minutes. NaCNBH₃ (0.043 g, 0.68 mmol) was added to the reaction and itwas monitored by HPLC and LC/MS. The reaction was shown to be completein 3 hours and it was quenched with MeOH (30 mL). The final product wasisolated by preparative HPLC in 10% yield as a yellow solid. ESI-MS: m/z(M+H) 601.

Synthesis of 7-Aminomethyl Doxycycline

To 1 gram of 9-tert-butyl-doxycycline, dissolved in 15 ml ofmethanesulfonic acid, was added an excess of HMBC(Hydroxymethyl-carbamic acid benzyl ester). The reaction mixture wasmonitored by analytical HPLC. The LCMS showed MS: 530 corresponding tothe desired material, 7-aminomethyl-9-t-butyl doxycycline. The productwas isolated via preparative HPLC and the structure confirmed by NMR.Removal of the t-butyl in triflic acid afforded the 7-aminomethyldoxycycline in good yield.

Synthesis of 9-(3′,3′,3′-Trifluoropropylamino)methyl Minocycline

9-formyl-minocycline (0.2 g, 0.42 mmol) was combined with InCl₃ (0.01 g,0.005 mmol), 3,3,3-trifluoropropylamine.HCl (0.25 g, 1.7 mmol), Et₃N(0.17 g, 1.7 mmol), and DMF (10 mL) in a glass vial. The reaction wasstirred at room temperature under argon for 1 hour. NaCNBH₃ (0.032 g,0.50 mmol) was added to reaction solution and was monitored by HPLC andLC/MS. The reaction was complete within 1 hour, quenched with MeOH (20mL) and the solvent evacuated in vacuo. The final product was isolatedby preparative HPLC in 25% yield as a yellow solid. ESI-MS: m/z (M+H)583.

9-(4′-Difluoromethylene-N-piperidinyl) methyl Minocycline

Anhydrous tetrahydrofuran (THF, 200 mL) was placed in a flame-dried 500mL round bottom flask at 0° C. in an ice bath. Dibromodifluoromethane(97%, Aldrich, 10.00 mL, 106.19 mmol, 4.3 eq.) was added via syringe.Ten minutes later, Hexamethylphosphorous triamide (HMPT, 97%, Aldrich,19.50 mL, 104.07 mmol, 4.2 eq.) was added dropwise. The clear solutionturned milky white and was stirred for 1 hour at 0° C. A solution oftert-Butyl 4-oxo-1-piperidinecarboxylate (98%, Aldrich, 5.00 g, 24.59mmol, 1.0 eq.) in anhydrous THF (50 mL) was then added dropwise viasyringe at 0° C. and the solution was allowed to warm up slowly to roomtemperature over 1 hour by removing the ice bath. The powdered zinc(99.998%, Aldrich, powdered, −100 mesh, 6.56 g, 98.34 mmol, 4.0 eq.) wasthen added followed by HMPT (1.15 mL, 6.14 mmol, 25%) and the reactionmixture was refluxed for 3 hours. Water (250 mL) and Diethyl ether(Et₂O, 250 mL) were added and the mixture was extracted with Et₂O (3times 100 mL). The combined organic layers were washed with a saturatedsolution of Copper(II) sulfate (CuSO₄) in Water (150 mL) then with water(150 mL). The organic layer was dried over Magnesium sulfate (MgSO₄),filtered, and evaporated under reduced pressure to yield the desiredfluorinated piperidine as a yellow oil, which was used without furtherpurification in the next step.

A 100 mL round bottom flask equipped with a magnetic stirring bar wasloaded with the BOC-protected piperidine (2.00 g, 8.57 mmol, 1.0 eq.) ina saturated HCl solution in Methanol (50 mL) at room temperature. Themixture was then stirred at 40° C. for 30 minutes and the solvent wasevaporated under reduced pressure to a minimal volume. The HCl salt wasthen precipitated from Et₂O, filtered, and dried in vacuo to yield thedesired fluorinated piperidine (1.10 g, 6.49 mmol, 76% yield) as a beigesolid used without further purification in the next step.

A flame-dried 50 mL round bottom flask equipped with a magnetic stirringbar was loaded with 9-Formyl-minocycline (500 mg, 1.03 mmol, 1.0 eq.) inanhydrous Dimethylformamide (DMF, 10.00 mL) at room temperature. Indiumchloride (InCl₃, 99.999%, Aldrich, 59 mg, 0.27 mmol, 26%) was added andthe reaction mixture was stirred at 30° C. for 10 minutes. The amine(350 mg, 2.06 mmol, 2.0 eq.) was added in anhydrous DMF (2 mL), followedby Triethylamine (NEt₃, 99.5%, Alfa-Aesar, 290 μL, 2.08 mmol, 2.0 eq.).The mixture was then stirred at 30° C. for 1 hour and Sodiumtriacetoxyborohydride (NaBH(OAc)₃, 95%, Aldrich, 220 mg, 1.04 mmol, 1.0eq.) was added followed by more NEt₃ (300 μL). After 2 hours, thereaction was done and the solvent evaporated under reduced pressure. Theresidue was purified by preparative HPLC (Acetonitrile/Water/0.1%Trifluoroacetic acid gradient) to yield the desired product as a yellowsolid. MS m/z 603.

Synthesis of 9-(4′-Fluoro-N-Piperdinyl) methyl Doxycycline

The compound was prepared from Doxycycline (2.5 g, 5.0 mmol) dissolvedin MeOH (anhydrous) (25 mL) and combined with AgSO4 (3.7 g, 11 mmol) andI₂ (3.1 g, 11 mmol) in a 100 mL round bottom flask. H₂SO_(4conc) (2drops) was added to the reaction solution and stirred at roomtemperature under argon for 1 hour. The reaction solution turned brightyellow after 30 minutes and the reaction was monitored by LC/MS andshown to be complete in 1 hour. Sodium sulfite (sat) (8 mL) was added tothe reaction solution and a thick yellow precipitate was formed. Themixture was stirred at room temperature for 20 minutes. The mixture wasdiluted with CH₃CN (75 mL), filtered through celite and evaporatedsolvent in vacuo to yield 1.7 g of crude 9-iodo-doxycycline material.

9-iodo-doxycycline (1.3 g, 2.4 mmol) was dissolved in anhydrous DMF (20mL) in a 200 mL 2 neck round bottom flask and Pd(PPh₃)₄ (0.82 g, 0.71mmol) was added. A CO-filled balloon was placed on top neck of reactionflask and CO was bubbled directly into reaction from lecture bottole.The flask was then opened to the balloon and SnBu₃H (0.70 g, 2.7 mmol)was added via syringe pump over 1 hour. The reaction solution was heatedto 65° C. during the tin addition. The reaction was monitored by LC/MSand it was shown to be complete once the tin addition was complete.Water (0.1% TFA) (200 mL) was then added to reaction solution and ayellow precipitate formed. The mixture was then filtered through celiteand the filtrate was evaporated in vacuo. A brown/yellow solid in 50%yield was isolated.

(9-formyl-doxycycline (0.20 g, 0.42 mmol) combined with InCl₃ (0.01 g,0.042 mmol), 4-fluoropiperidine (0.13 g, 0.84 mmol), Et₃N (0.09 g, 0.84mmol), and DMF (5 mL) in a glass vial. The mixture was stirred underargon at room temperature for 30 minutes. NaCNBH₃ (0.037 g, 0.59 mmol)was added to the reaction vial and the reaction continued to be stirredat room temperature under argon. The reaction was monitored by LC/MS andHPLC and shown to be complete after 1 hour. The reaction was quenchedwith MeOH (15 mL) and the solvent was evacuated in vacuo. The productwas isolated by preparative HPLC in 10% yield as a yellow solid. ESI-MS:m/z (M+H) 559.

Synthesis of 9-(Benzyl-methyl-amino)-Propynyl)-Minocycline

7-Iodo-minocycline (1.08 g, 1.86 mmol), taken in 25 mL of acetonitrilewas degassed and purged with nitrogen (three times). To this suspensionPd(OAc)₂ (20 mg, 0.089 mmol), CuI (10 mg, 0.053 mmol), (o-tolyl)₃P (56mg, 0.186 mmol) were added and purged with nitrogen for few minutes.Benzyl-methyl-prop-2-ynyl-amine (318 μL, 2 mmol) and triethylamine (1mL) were added to the suspension. It turned into a brown solution afterthe addition of Et₃N. The reaction mixture was then heated to 70 C for 2hours. The progress of the reaction was monitored by HPLC/LCMS. It wasthen cooled down to room temperature and was filtered through celite.Evaporation of the solvent gave a brown solid, which was then purifiedon preparative HPLC to afford the desired compound. LC-MS (M+1 615).

Synthesis of 8-(2′-[(2′-Fluoro-ethylamino)-methyl]-phenyl)-Sancycline

Step 1:

To a stirred solution (cooled at 0° C., ice-bath) of 9-amino-sancycline(7 g, 16.3 mmol) in 200 mL of MeOH, 48% HBF₄ solution (5.32 mL, 40.75mmol) was added slowly under an argon atmosphere. After 5 minutes,n-BuNO2 (2.1 mL, 17.93 mmol) was added slowly (dropwise). The reactionmixture was then stirred at 0 C for 3 hours (monitored by HPLC/LC-MS).NaN₃ (1.06 g, 16.3 mmol) was then added the reaction mixture (all atonce). The reaction mixture was stirred at 0 C for another 3 hours(monitored by HPLC/LC-MS). The reaction mixture was then poured slowlyinto stirring diethyl ether (˜1 L at ice-bath temperature). A yellowprecipitate was obtained and it was filtered, washed with ether (20ml×3) and dried under vacuum, sealed in a vial and stored at 0 C.Isolated yield 7 g.

Step 2:

Hydrobromic acid (30% in acetic acid) (14 mL) was added to a flask andcooled to 0 C. 9-Azido-sancycline (1 g, 2.2 mmol) was added to the flaskand the reaction was left to stir for one hour. After 1 hour, thereaction was complete. The reaction mixture was precipitated in 300 mLof diethyl ether. After letting the solution settle, the top layer ofdiethyl ether was decanted and the reaction mixture was dried undervacuum. A brown-black solid was then dissolved in methanol andprecipitated using diethyl ether. The solid obtained was filtered anddried under vacuum.

Step 3:

To a stirred solution (cooled at 0 C, ice-bath) of8-bromo-9-amino-sancycline (828 mg, 1.6 mmol) in 200 mL of MeOH, 48%HBF₄ solution (0.53 mL, 4.0 mmol) was added slowly under an argonatmosphere. After 5 minutes, n-BuNO₂ (0.2 mL, 1.79 mmol) was addedslowly (dropwise). The reaction mixture was then stirred at 0 C for 2hours and left overnight at room temperature (monitored by HPLC/LC-MS).The solvent was evaporated and the crude material obtained wasprecipitated using diethyl ether (300 mL). The solid obtained wasfiltered and dried under vacuum.

Step 4:

8-Bromo-sancycline (492 mg, 1 mmol) and Pd(OAc)₂ (22 mg, 0.1 mmol) weretaken in methanol (150 mL) and purged with argon while heating thereaction mixture at 65 C (oil bath temperature). After 10 minutes, anaqueous solution of sodium carbonate (315 mg, 3 mmol in 10 mL of water)was added. A yellow precipitate was obtained which was further heatedfor another 10 minutes, before adding a DMF solution of the boronic acid(300 mg, 2 mmol in 10 mL of DMF). The reaction was then heated at 65 Cfor 3 hours. The reaction was monitored by HPLC/LCMS. The mixture wascooled down to room temperature and then filtered through celite. Thesolvent was then evaporated and the crude material obtained wasprecipitated using methanol/diethyl ether (10/200 mL). The crudematerial was then filtered and dried under vacuum. The yellow-brownmaterial obtained was used as such without further purification.

Step 5:

To a solution of 8-(2-formyl-phenyl)-sancycline (518 mg, 1 mmol) in 30mL of DCE under an argon atmosphere, 2-fluoro-ethylamine hydrochloride(198 mg, 2 mmol) and triethylamine (202 μL, 2 mmol) were added. Thereaction mixture was then stirred at room temperature for 2 hours. Thereaction was monitored by using HPLC/LCMS, and was completed in 2 hours.The solvent was then evaporated and the crude material was purifiedusing preparative HPLC to afford the desired compound. LC-MS (M+1 566).

7-Pyrazolyl-Sancycline

To a stirred solution of 7-Iodo sancycline (100 mg, 0.153 mmol) in DMF(1 mL) was added pyrozole-4-boronic acid pinacole cyclic ester (77 mg,0.40 mmol), methanol (1.5 mL), tetrakis(triphenylphosphine)palladium (18mg, 0.015 mmol) and a solution containing 250 mg CsCO₃ in 0.7 mL water.The reaction mixture was then subject to microwave irradiation at atemperature of 100 C for 5 minutes. The reaction was then diluted with100 mL of water and TFA was used to lower the pH to 2. This solution wasthen filtered through celite, and loaded onto a plug of divinyl benzeneresin (DVB). The plug containing the product was washed with water (200mL) before the final compound was eluted with MeCN and reduced by rotaryevaporation. The crude material was purified by reverse phase HPLC togive the final product (64 mg, 0.12 mmol, 75% yield) LCMS m/z=481.2115(M+H).

Synthesis of 9-[(2,2,2-Trifluoro-ethyl)-hydrazonomethyl]-Minocycline

To a solution of 9-formyl minocycline (485 mg, 1 mmol) in 30 mL of DMFunder an argon atmosphere, indium trichloride (22 mg, 0.1 mmol) andtrifiuoroethylhydrazine (228 μL, 2 mmol) were added. The reactionmixture was then stirred at room temperature for 30 minutes. Thereaction was monitored by using HPLC/LCMS, and was completed in 30minutes. The solvent was then evaporated and the crude material waspurified using preparative HPLC to afford the desired compound. LC-MS(M+1 582).

Synthesis of 9-(1′-Isopropyl-4′-piperidinyl) amino Sancycline

To a solution of 9-amino sancycline HCl salt (0.5 g, 1 mmol) in 40 ml ofmethanol and was added 1-isopropyl-4-piperidone (0.14 g, 2 mmol). Thesolution was stirred for 5 minutes at room temperature. Sodiumcyanoborohydride (62.5 mg, 1 mmol) was introduced, followed by theaddition of 4 ml of AcOH. The mixture was stirred at room temperaturefor 1 hour until all starting material disappeared. The suspension wasfiltered and purified by HPLC to afford the title compound (210 mg).LC-MS (M+1 555).

Synthesis of 9-(3-t-butyl-N-imidazolyl)-methyl)-Minocycline

To a stirred solution of 9-aminomethyl-minocycline (2.50 g, 4.14 mmol)in DMF (25 mL) and MeOH (15 mL) was added 1-bromopinacolone (1.34 mL,1.01 g, 5.63 mmol) and Cs₂CO₃ (5.0 mL of a 1N aqueous solution, 5.0mmol). The reaction was heated to 100° C. for 15 minutes in a pressurevesicle using microwave irradiation. The contents were then diluted withwater (1.0 L) and Na₂CO₃ was used to adjust the pH to 6. This solutionwas then filtered through celite and loaded onto a plug of divinylbenzene resin. The product was washed with water (500 mL) before it waseluted with MeCN and reduced by rotary evaporation. The crude materialwas purified by reverse phase HPLC to give the tert-butyl-ketoneintermediate (680 mg, 1.90 mmol, 50% yield). To a stirred solution ofthe tert-butyl-ketone intermediate (68 mg, 0.190 mmol) in formamide (1.0mL) was added triethyl-amine (0.020 mL, 28 mg, 0.27 mmol) to adjust thepH to 8. The reaction was heated to 100° C. for 5 minutes in a pressurevesicle using microwave irradiation. The contents were then diluted withwater (100 mL) and TFA was used to adjust the pH to 2. This solution wasthen filtered through celite, and loaded onto a plug of divinyl benzeneresin. The product was washed with water (200 mL) before it was elutedwith MeCN and reduced by rotary evaporation. The crude material waspurified by reverse phase HPLC to give the final compound (6.0 mg, 10μmol, 4% yield) LCMS m/z=594.4863 (M+H).

Synthesis of 9-(2-thiol 5-methyl-N-imidazolyl)-methyl Minocycline

To a stirred solution of 9-aminomethyl-minocycline (2.00 g, 4.12 mmol)in DMF (12 mL), MeOH (6.0 mL) and acetic acid (3.0 mL) was added KSCN(0.400 g, 4.12 mmol) and Acetol 0.400 mL, 0.370 g, 5.00 mmol). Thereaction was heated to 100° C. for 15 minutes in a pressure vesicleusing microwave irradiation. The contents were then diluted with water(1.0 L) and Na₂CO₃ was used to adjust the pH to 6. This solution wasthen filtered through celite and loaded onto a plug of divinyl benzeneresin. The product was washed with water (500 mL) before it was elutedwith MeCN and reduced by rotary evaporation. The crude material waspurified by reverse phase HPLC to give the final product (620 mg, 1.06mmol, 26% yield) LCMS m/z=584.3998 (M+H).

Synthesis of 7-(2′,2′-dimethyl-propyl)amino methyl Sancycline

1 g of 7-aminomethyl-sancycline, 3 equivalents of trimethylacetaldehydeand one equivalent of indium trichloride were dissolved in 10 ml of DMF.The mixture was stirred at room temperature for 15 minutes. To thismixture was added 3 equivalents of sodium triacetoxyborohydride. Theresulting reaction mixture was left stirring for several hours. Thereaction was monitored by analytical HPLC. The LCMS showed MS: 514 whichcorresponds to the desired material. The product was isolated viapreparative HPLC and the structure was confirmed by NMR.

Synthesis of 9-(Benzoimidazolyl)-Minocycline

To a stirred solution of the trifluoroacetic acid (TFA) salt of 9-formylminocycline (488 mg, 1.47 mmol) in DMF (3 mL) and MeOH (2 mL) was added1,2-phenylenediamine (80 mg, 0.74 mmol). The reaction was heated to 50 Cand was complete in 5 minutes. The contents were then diluted with water(500 mL) and TFA was used to adjust the pH to 2. This solution was thenfiltered through celite, and loaded onto a plug of divinyl benzeneresin. The plug containing the product was washed with water (300 mL)before it was eluted with MeCN and reduced by rotary evaporation. Thecrude material was purified by reverse phase HPLC to give theBenzoimidazol product (100 mg, 0.175 mmol, 10% yield) LCMS m/z=574.3637(M+H).

Example 2: In Vitro Minimum Inhibitory Concentration (MIC) Assay

The following assay is used to determine the efficacy of thetetracycline compounds against common bacteria. 2 mg of each compound isdissolved in 100 μl of DMSO. The solution is then added tocation-adjusted Mueller Hinton broth (CAMHB), which results in a finalcompound concentration of 200 μg per ml. The tetracycline compoundsolutions are diluted to 50 μl volumes, with a test compoundconcentration of 0.098 μg/ml. Optical density (OD) determinations aremade from fresh log-phase broth cultures of the test strains. Dilutionsare made to achieve a final cell density of 1×10⁶ CFU/ml. At OD=1, celldensities for different genera should be approximately:

E. coli 1 × 10⁹ CFU/ml S. aureus 5 × 10⁸ CFU/ml Enterococcus sp. 2.5 ×10⁹ CFU/ml

50 μl of the cell suspensions are added to each well of microtiterplates. The final cell density should be approximately 5×10⁵ CFU/ml.These plates are incubated at 35° C. in an ambient air incubator forapproximately 18 hr. The plates are read with a microplate reader andare visually inspected when necessary. The MIC is defined as the lowestconcentration of the tetracycline compound that inhibits growth.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, patents, and patentapplications cited throughout this application are hereby incorporatedby reference. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for thepresent invention and embodiments thereof.

1-49. (canceled)
 50. A method for ameliorating a tetracycline responsive state in a subject in need thereof, the method comprising administering to said subject a tetracycline compound of the following structural formula:

or a pharmaceutically acceptable salt thereof, such that said tetracycline responsive state in said subject is ameliorated.
 51. The method of claim 50, wherein said tetracycline responsive state is an inflammatory disorder.
 52. The method of claim 51, wherein said tetracycline responsive state is an inflammatory condition of the skin.
 53. The method of claim 51, wherein said tetracycline responsive state is an inflammatory disorder caused by trauma.
 54. The method of claim 51, wherein said tetracycline responsive state is an inflammatory disorder caused by radiation.
 55. The method of claim 50, wherein said subject is a human.
 56. A method for ameliorating a tetracycline responsive state in a subject in need thereof, the method comprising administering to said subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a tetracycline compound of the following structural formula:

or a pharmaceutically acceptable salt thereof, such that said tetracycline responsive state in said subject is ameliorated.
 57. The method of claim 56, wherein said tetracycline responsive state is an inflammatory disorder.
 58. The method of claim 57, wherein said tetracycline responsive state is an inflammatory condition of the skin.
 59. The method of claim 57, wherein said tetracycline responsive state is an inflammatory disorder caused by trauma.
 60. The method of claim 57, wherein said tetracycline responsive state is an inflammatory disorder caused by radiation.
 61. The method of claim 56, wherein said subject is a human. 